( K , L ) ISG qRT - PCR ( K ) and infection data ( L ) from primary monocyte - derived macrophages ( MDM ) infected for 24 h with LPV - treated HIV - 1 R9 BaL viruses ( 0 . 2 U RT / ml ) . Data are collated from two donors ( represented by circles and squares ) , n = 3 . Horizontal line represents the median . For experiments in which the virus dose used was normalised by RT activity , the number of genome copies was also measured by qPCR of virus . This gave dose equivalents of within 2 - 3 fold of RT equivalents .

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(K, L) ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDM) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n=3. Horizontal line represents the median. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within 2-3 fold of RT equivalents.


( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) with varying proportions of ΔCA - SP1 protease cleavage mutation detecting p24 .

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(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) with varying proportions of ΔCA-SP1 protease cleavage mutation detecting p24.


( B ) Titration of HIV - 1 GFP ΔCA - SP1 viruses on U87 cells . Mean ± SD , n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Titration of HIV-1 GFP ΔCA-SP1 viruses on U87 cells. Mean ± SD, n=3.


( C ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


D ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

D) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . E ) CXCL - 10 protein in supernatants from ( C , D ) ( ELISA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). E) CXCL-10 protein in supernatants from (C, D) (ELISA).


( F ) RT products from THP - 1 cells transduced for 24 h with 6 × 109 genomes / ml ( approx . 0 . 5 U RT / ml ) HIV - 1 GFP ΔCA - SP1 viruses .

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(F) RT products from THP-1 cells transduced for 24 h with 6×109 genomes/ml (approx. 0.5 U RT/ml) HIV-1 GFP ΔCA-SP1 viruses.


( G ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 016 - 0 . 2 U RT / ml ) . Data are shown as individual measurements , representative of 2 repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.016 - 0.2 U RT/ml). Data are shown as individual measurements, representative of 2 repeats.


( H ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 mutant , or stimulated with 4 μg / ml cGAMP as a control , in the presence of DMSO vehicle or 2 μM ruxolitinib .

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(H) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 mutant, or stimulated with 4 μg/ml cGAMP as a control, in the presence of DMSO vehicle or 2 μM ruxolitinib.


( I ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 3 × 108 genomes / ml or 1 . 5 × 109 genomes / ml , equivalent to 0 . 1 U RT / ml and 0 . 5 U RT / ml ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (3×108 genomes/ml or 1.5×109 genomes/ml, equivalent to 0.1 U RT/ml and 0.5 U RT/ml).


( J ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib .

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(J) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib.


primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib . K ) CXCL - 10 protein in supernatants from ( J ) ( ELISA ) .

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primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib. K) CXCL-10 protein in supernatants from (J) (ELISA).


( A ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O

(A) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.


THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . B ) CXCL - 10 protein in supernatants from ( A ) ( ELISA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir. B) CXCL-10 protein in supernatants from (A) (ELISA).


ISG qRT - PCR from THP - 1 - IFIT - 1 cells transduced for 24 h with 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 109 and 3 × 109 genomes / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir .

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ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.


( E ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ΔCA - SP1 ( WT ) , 75 % ΔCA - SP1 , 75 % ΔCA - SP1 carrying a mutation in reverse transcriptase ( 75 % ΔCA - SP1 RT D185E ) or 75 % ΔCA - SP1 carrying a mutation in integrase ( 75 % ΔCA - SP1 INT D116N ) ( 3 . 75 × 109 , 7 . 5 × 109 and 1 . 5 × 1010 genomes / ml ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % ΔCA-SP1 (WT), 75 % ΔCA-SP1, 75 % ΔCA-SP1 carrying a mutation in reverse transcriptase (75 % ΔCA-SP1 RT D185E) or 75 % ΔCA-SP1 carrying a mutation in integrase (75 % ΔCA-SP1 INT D116N) (3.75×109, 7.5×109 and 1.5×1010 genomes/ml).


( F ) CXCL - 10 protein in supernatants from ( E ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

(F) CXCL-10 protein in supernatants from (E) (ELISA). Triangles indicate CXCL-10 not detected.


( A ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING or MAVS , or a gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP 75 % ΔCA - SP1 ( 0 . 4 U RT / ml ) or stimulated by transfection with either HT - DNA ( 0 . 1 μg / ml ) or poly I : C ( 0 . 5 μg / ml ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

(A) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING or MAVS, or a gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP 75 % ΔCA-SP1 (0.4 U RT/ml) or stimulated by transfection with either HT-DNA (0.1 μg/ml) or poly I:C (0.5 μg/ml).


IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line stimulated for 24 h with poly I : C ( transfection , 0 . 5 μg / ml ) , HT - DNA ( transfection , 0 . 1 μg / ml ) , LPS ( 50 ng / ml ) or cGAMP ( transfection , 0 . 5 μg / ml )

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IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line stimulated for 24 h with poly I:C (transfection, 0.5 μg/ml), HT-DNA (transfection, 0.1 μg/ml), LPS (50 ng/ml) or cGAMP (transfection, 0.5 μg/ml)


IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml)


PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml ) ( C ) . D ) CXCL - 10 protein in supernatants from ( C ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml) (C). D) CXCL-10 protein in supernatants from (C) (ELISA). Triangles indicate CXCL-10 not detected.


( E ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) .

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(E) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).


( F ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(F) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).


PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . G ) CXCL - 10 protein in supernatants from ( E ) .

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PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). G) CXCL-10 protein in supernatants from (E).


PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . ( H ) CXCL - 10 protein in supernatants from ( F ) ( ELISA ) .

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PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). (H) CXCL-10 protein in supernatants from (F) (ELISA).


( I ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 control cells transduced for 48 h with WT , DRV - treated ( DRV , 12 . 5 nM ) or HIV - 1 GFP containing 90 % ΔCA - SP1 ( 1 × 1010 genomes / ml ) in the presence of DMSO vehicle , 2 μM ruxolitinib or 0 . 5 μg / ml H151 .

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(I) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 control cells transduced for 48 h with WT, DRV-treated (DRV, 12.5 nM) or HIV-1 GFP containing 90 % ΔCA-SP1 (1×1010 genomes/ml) in the presence of DMSO vehicle, 2 μM ruxolitinib or 0.5 μg/ml H151.


Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with a fixed dose of HIV - 1 GFP ( 5 × 107 genomes / ml ) and increasing doses of HIV - LUC ΔCA - SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with a fixed dose of HIV-1 GFP (5×107 genomes/ml) and increasing doses of HIV-LUC ΔCA-SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.


Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with LPV - treated HIV - LUC viruses Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with LPV-treated HIV-LUC viruses Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.


( A ) Infection data for THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(A) Infection data for THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).


THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . B ) IFIT - 1 reporter activity in supernatant from ( A ) .

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THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM). B) IFIT-1 reporter activity in supernatant from (A).


ISG qRT - PCR from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 25 - 1 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).


( E ) CXCL - 10 protein in supernatants of THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(E) CXCL-10 protein in supernatants of THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).


( F ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) and ruxolitinib ( Rux , 2 μM ) as indicated .

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(F) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM) and ruxolitinib (Rux, 2 μM) as indicated.


( G ) IRF reporter activity from THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP ( 3 U / ml and 6 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(G) IRF reporter activity from THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).


( H ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells lacking MAVS or a matching gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP ( 3 U / ml and 6 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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(H) IFIT-1 reporter activity from THP-1-IFIT-1 cells lacking MAVS or a matching gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).


Substrate was synthesized in reticulocyte lysate in the presence of 35S - methionine and mixed with SecA and SecYEG proteoliposomes . After incubation with ATP , the samples were treated with proteinase K to degrade any non - translocated material . Where indicated , the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X - 100 ( TX - 100 ) . Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT , generating protease - protected full - length ( FL ) substrate or a translocation intermediate ( IM ) , respectively . All samples were analyzed by SDS PAGE followed by autoradiography . Lane 1 shows 10 % of the input sample . The gel is representative of three replicates . Molecular weight markers are estimated from Coomassie - stained gels run in parallel .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Substrate was synthesized in reticulocyte lysate in the presence of 35S-methionine and mixed with SecA and SecYEG proteoliposomes. After incubation with ATP, the samples were treated with proteinase K to degrade any non-translocated material. Where indicated, the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X-100 (TX-100). Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT, generating protease-protected full-length (FL) substrate or a translocation intermediate (IM), respectively. All samples were analyzed by SDS PAGE followed by autoradiography. Lane 1 shows 10% of the input sample. The gel is representative of three replicates. Molecular weight markers are estimated from Coomassie-stained gels run in parallel.


Translocation intermediates ( IM ) were incubated in the presence of different nucleotides ( Nuc . ) for different time periods and then treated with proteinase K ( Prot . K ) . One sample received hexokinase and glucose ( HKG ) to generate ADP , other samples ADP • BeFx or ATPγS . The gel is representative of two replicates . Quantification of ( C ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show the average of replicates . Colors correspond to different nucleotides as shown in the inset box . As in ( C ) , but DTT was added concurrently with HKG or nucleotides . The gel is representative of two replicates . Quantification of ( E ) as in ( D ) , except intensities were calculated for the FL bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Translocation intermediates (IM) were incubated in the presence of different nucleotides (Nuc.) for different time periods and then treated with proteinase K (Prot. K). One sample received hexokinase and glucose (HKG) to generate ADP, other samples ADP•BeFx or ATPγS. The gel is representative of two replicates.   Quantification of (C). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show the average of replicates. Colors correspond to different nucleotides as shown in the inset box.   As in (C), but DTT was added concurrently with HKG or nucleotides. The gel is representative of two replicates.   Quantification of (E) as in (D), except intensities were calculated for the FL bands.


WB SecA with additional mutations ( WT , Y794G , R342E ) was added to preformed intermediates ( IM ) and incubated for the indicated times before proteolysis . The gel is representative of two replicates . Quantification of ( B ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to WB SecA carrying additional mutations , as shown in the inset box .

WB SecA with additional mutations (WT, Y794G, R342E) was added to preformed intermediates (IM) and incubated for the indicated times before proteolysis. The gel is representative of two replicates.   Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to WB SecA carrying additional mutations, as shown in the inset box.


Imidazole ( imi ) and either WT or mutant SecA were added to translocation intermediates ( IM ) pre - formed with SecA dN20 : : H6 . The samples were incubated for the indicated times before proteolysis with proteinase K ( Prot . K ) . The gel is representative of two replicates , except for the experiment with the R792G / K797G mutant and the 30 - min time points . Quantification of ( B ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to different SecA mutants as shown in the inset box . As in ( B ) , but ATPγS was added together with imidazole . The gel is representative of two replicates . Quantification of ( E ) as in ( C ) . As in ( D ) , but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA . The gel is representative of two replicates . Quantification of ( G ) as in ( F ) , except that both the intensities of the IM ( darker colors ) and FL ( lighter colors ) bands were measured .

B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Imidazole (imi) and either WT or mutant SecA were added to translocation intermediates (IM) pre-formed with SecA dN20::H6. The samples were incubated for the indicated times before proteolysis with proteinase K (Prot. K). The gel is representative of two replicates, except for the experiment with the R792G/K797G mutant and the 30-min time points. Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to different SecA mutants as shown in the inset box. As in (B), but ATPγS was added together with imidazole. The gel is representative of two replicates. Quantification of (E) as in (C). As in (D), but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA. The gel is representative of two replicates.   Quantification of (G) as in (F), except that both the intensities of the IM (darker colors) and FL (lighter colors) bands were measured.


Time - lapse imaging of an oocyte expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints of four stages from anaphase I onset to CSF - dependent MII arrest . The 3D positions of centromeres are shown as black dots in a side view ( along the spindle equator , middle row ) and top view ( perpendicular to the equator plane , bottom row ) . Tracks of individual centromeres are color - coded according to the speed , as indicated by the colour bar . Time is shown after anaphase I onset ( min : sec ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-lapse imaging of an oocyte expressing CENP-C-EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints of four stages from anaphase I onset to CSF-dependent MII arrest. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown after anaphase I onset (min:sec). Scale bars, 5 μm.


Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . On the schemes above the charts in ( A ) and ( C ) , chromosomes are red with green centromeres . Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1 . The black arrow on the scheme above the chart indicates chromosome - centre distance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1. The black arrow on the scheme above the chart indicates chromosome-centre distance.


Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . Changes in the speed of all chromosomes observed for the oocyte from Fig 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. Changes in the speed of all chromosomes observed for the oocyte from Fig 1.


Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . On the schemes above the charts in ( A ) and ( C ) , chromosomes are red with green centromeres . ( C ) Changes in inter - centromere distance ( indicated on a scheme by the black arrow ) , observed for the oocyte from Fig 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. (C) Changes in inter-centromere distance (indicated on a scheme by the black arrow), observed for the oocyte from Fig 1.


Kinetochore - MT attachments at the prometaphase II stage were visualized using an anti - tubulin antibody ( white ) , an anti - centromeric ACA antibody ( green ) and DAPI to label chromatin ( red ) . The image represents maximum intensity projection through all z - planes containing MTs . Representative chromosomes displaying syntelic , amphitelic or merotelic / lateral attachments are enclosed in white frames and their enlarged single z - plane images are shown to the right . PB : polar body . Scale bar , 10μm . Chromosomes with syntelic ( red ) , amphitelic ( green ) and merotelic / lateral ( grey ) attachments were plotted according to their inter - centromere distance ( on the horizontal axis ) and distance to spindle equator ( vertical axis ) . Data shown for 156 chromosomes taken from 8 prometaphase II oocytes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kinetochore-MT attachments at the prometaphase II stage were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maximum intensity projection through all z-planes containing MTs. Representative chromosomes displaying syntelic, amphitelic or merotelic/lateral attachments are enclosed in white frames and their enlarged single z-plane images are shown to the right. PB: polar body. Scale bar, 10μm. Chromosomes with syntelic (red), amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). Data shown for 156 chromosomes taken from 8 prometaphase II oocytes.


Time - lapse imaging of a representative MII oocyte with a normal segregation pattern ( i . e . all 20 chromosomes show synchronous equational chromatid separation at anaphase onset , leaving no chromatids behind at the midzone ) , expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II . Number next to the circles enclosing segregating chromatids denotes amount of chromatids segregated to each of the poles . The 3D positions of centromeres are shown as black dots in a side view ( along the spindle equator , middle row ) and top view ( perpendicular to the equator plane , bottom row ) . Tracks of individual centromeres are color - coded according to their speed , as indicated by the colour bar . Time is shown relative to anaphase II onset ( min : sec ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-lapse imaging of a representative MII oocyte with a normal segregation pattern (i.e. all 20 chromosomes show synchronous equational chromatid separation at anaphase onset, leaving no chromatids behind at the midzone), expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. Number next to the circles enclosing segregating chromatids denotes amount of chromatids segregated to each of the poles. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to their speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.


Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A - D on the vertical axis , and time in min on the horizontal axis relative to the anaphase II onset . Thin grey lines in A - D represent individual chromosomes ; thick black line in C - D shows the mean value for each timepoint . On the schemes near the charts chromosomes are red with green centromeres ; spindle equator in A and spindle axis in B - C are indicated by dashed lines . Changes in distance to the equator plane for all chromosomes in the representative oocyte with normal segregation pattern , shown in Fig 3 . The analysed distance is indicated by a black arrowed line on the scheme above the chart . Changes in distance to the spindle axis ( shown on the scheme by a black arrowed line ) for all chromosomes in oocyte from Fig 3 . Changes in chromosome orientation for all chromosomes in oocyte from Fig 3 . The orientation is characterized by the angle ( highlighted black ) between the spindle axis and the line connecting the sister centromeres ( black solid line on the scheme ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by dashed lines. Changes in distance to the equator plane for all chromosomes in the representative oocyte with normal segregation pattern, shown in Fig 3. The analysed distance is indicated by a black arrowed line on the scheme above the chart. Changes in distance to the spindle axis (shown on the scheme by a black arrowed line) for all chromosomes in oocyte from Fig 3. Changes in chromosome orientation for all chromosomes in oocyte from Fig 3. The orientation is characterized by the angle (highlighted black) between the spindle axis and the line connecting the sister centromeres (black solid line on the scheme).


Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A - D on the vertical axis , and time in min on the horizontal axis relative to the anaphase II onset . Thin grey lines in A - D represent individual chromosomes ; thick black line in C - D shows the mean value for each timepoint . ( D ) Changes in inter - centromere distance , shown on the scheme by black arrowed line , for all chromosomes in oocyte from Fig 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. (D) Changes in inter-centromere distance, shown on the scheme by black arrowed line, for all chromosomes in oocyte from Fig 3.


Kinetochore - MT attachments in oocytes released from the CSF - dependent metaphase arrest were visualized using an anti - tubulin antibody ( white ) , an anti - centromeric ACA antibody ( green ) and DAPI to label chromatin ( red ) . The image represents maxiumum intensity projection through all z - planes containing MTs . Single z - planes of representative chromosomes displaying amphitelic or merotelic / lateral attachments are shown to the right . Scale bar , 10μm . Chromosomes with amphitelic ( green ) and merotelic / lateral ( grey ) attachments were plotted according to their inter - centromere distance ( on the horizontal axis ) and distance to spindle equator ( vertical axis ) . The inter - centromere distance in fixed oocytes is 1 . 2 ± 0 . 2 μm for amphitelic and 1 . 0 ± 0 . 2 μm for merotelic attachments ( Mean ± SD ) . Data shown for 95 chromosomes taken from 5 oocytes released from the CSF - dependent metaphase II arrest .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kinetochore-MT attachments in oocytes released from the CSF-dependent metaphase arrest were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maxiumum intensity projection through all z-planes containing MTs. Single z-planes of representative chromosomes displaying amphitelic or merotelic/lateral attachments are shown to the right. Scale bar, 10μm. Chromosomes with amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). The inter-centromere distance in fixed oocytes is 1.2±0.2 μm for amphitelic and 1.0±0.2 μm for merotelic attachments (Mean±SD). Data shown for 95 chromosomes taken from 5 oocytes released from the CSF-dependent metaphase II arrest.


A time - lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II . A lagging chromatid is indicated by a blue arrowhead . Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole . The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots , and centromeres of laggard - producing chromosome as red dots in a side view along the spindle equator . One of the centromeres is left behind at anaphase ( blue arrowhead ) , but will segregate equationally . Tracks of individual centromeres are color - coded according to the speed , as indicated by the colour bar . Time is shown relative to anaphase II onset ( min : sec ) . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A time-lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. A lagging chromatid is indicated by a blue arrowhead. Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole. The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots, and centromeres of laggard-producing chromosome as red dots in a side view along the spindle equator. One of the centromeres is left behind at anaphase (blue arrowhead), but will segregate equationally. Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.


Chromosome parameters obtained for laggard - producing chromosomes are shown in A - D on the vertical axis , time in min on the horizontal axis relative to the anaphase II onset . Light blue lines indicate 12 laggard - producing chromosomes with correct ( equational ) chromatid segregation ; darker line highlights one chromosome with chromatid non - disjunction . On the schemes near the charts chromosomes are red with green centromeres ; spindle equator in A and spindle axis in B - C are indicated by black dashed lines . Changes in the distance to the equator plane ( shown by a black arrowed line on the scheme ) for 13 laggard - producing chromosomes . All laggard - producing chromosomes are positioned above the equator at the start of analysis period . Black dotted lines indicate the average thickness of the metaphase plate . Changes in the distance to spindle axis ( shown by a black arrowed line on the scheme ) for 13 laggard - producing chromosomes . Black dotted line indicates the average radius of the metaphase plate . Changes in the angle to the spindle axis ( highlighted in black on the scheme ) for 13 laggard - producing chromosomes . Dashed line on the chart indicates the mean angle for normally segregating chromosomes . Changes in the inter - centromere distances ( shown by black arrowed line on the scheme ) for 13 laggard - producing chromosomes . Dashed line indicates the mean inter - centromere distance for normally segregating chromosomes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome parameters obtained for laggard-producing chromosomes are shown in A-D on the vertical axis, time in min on the horizontal axis relative to the anaphase II onset. Light blue lines indicate 12 laggard-producing chromosomes with correct (equational) chromatid segregation; darker line highlights one chromosome with chromatid non-disjunction. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by black dashed lines. Changes in the distance to the equator plane (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. All laggard-producing chromosomes are positioned above the equator at the start of analysis period. Black dotted lines indicate the average thickness of the metaphase plate. Changes in the distance to spindle axis (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. Black dotted line indicates the average radius of the metaphase plate. Changes in the angle to the spindle axis (highlighted in black on the scheme) for 13 laggard-producing chromosomes. Dashed line on the chart indicates the mean angle for normally segregating chromosomes. Changes in the inter-centromere distances (shown by black arrowed line on the scheme) for 13 laggard-producing chromosomes. Dashed line indicates the mean inter-centromere distance for normally segregating chromosomes.


B WT and SirT7 - / - mice at 10 days ( top ) and 2 months ( bottom ) of age .

O O O O O O O O O O O O O O O O O O O O O O O

B WT and SirT7-/- mice at 10 days (top) and 2 months (bottom) of age.


C Weight distribution of WT and SirT7 - / - mice ( n = 3 - 10 female mice per time point and genotype ; P value = 0 . 003 by unpaired t - Test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Weight distribution of WT and SirT7-/- mice (n=3-10 female mice per time point and genotype; P value = 0.003 by unpaired t-Test).


Kaplan Meier survival curves ( n = 170 WT and n = 58 SirT7 - / - mice ; Log - rank Test P value < 0 . 0001 ) .

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Kaplan Meier survival curves (n=170 WT and n=58 SirT7-/- mice; Log-rank Test P value <0.0001).


A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7 - / - mice compared with WT .

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A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7-/- mice compared with WT.


B Representative gonadal fat pads from WT and SirT7 - / - 16 month old mice .

B Representative gonadal fat pads from WT and SirT7-/- 16 month old mice.


C Quantitation of body weight ( left ) and gonadal fat pad mass normalized to total body weight ( right ) ( mean ± SEM ; 4 samples per genotype ) .

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C Quantitation of body weight (left) and gonadal fat pad mass normalized to total body weight (right) (mean ± SEM; 4 samples per genotype).


D IGF - 1 protein levels in serum measured by ELISA ( mean ± SEM ; 3 - 8 mice per genotype and age group ) .

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D IGF-1 protein levels in serum measured by ELISA (mean ± SEM; 3-8 mice per genotype and age group).


E , F Dot plots of Lin - Sca1 + cKit + ( LSK ) cells ( middle and right ) from WT and SirT7 - / - bone marrow cells . Cells were gated for negative staining of lineage markers B220 , CD3 , CD11b , CD19 , Gr - 1 , and Ter - 119 ( Left ) , and analyzed for Sca1 and cKit expression ( middle and right ) . ( F ) Quantitation of ( E ) ( mean ± SEM ; 4 mice per genotype ) .

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E, F Dot plots of Lin-Sca1+cKit+ (LSK) cells (middle and right) from WT and SirT7-/- bone marrow cells. Cells were gated for negative staining of lineage markers B220, CD3, CD11b, CD19, Gr-1, and Ter-119 (Left), and analyzed for Sca1 and cKit expression (middle and right). (F) Quantitation of (E) (mean ± SEM; 4 mice per genotype).


G Bone marrow , thymus , and spleen cell number in young WT and SirT7 - / - mice ( mean ± SEM ; 3 - 5 mice per genotype ) .

G Bone marrow, thymus, and spleen cell number in young WT and SirT7-/- mice (mean ± SEM; 3-5 mice per genotype).


H mRNA expression of p16 gene normalized to GAPDH measured by RT - PCR from young and old WT and SirT7 - / - fibroblasts ( mean ± SEM ; 3 samples per genotype ) .

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H mRNA expression of p16 gene normalized to GAPDH measured by RT-PCR from young and old WT and SirT7-/- fibroblasts (mean ± SEM; 3 samples per genotype).


I , J Senescence - associated β - galactosidasestaining ( I , blue ) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media . ( J ) Quantitation of the number of senescent cells shown in ( I ) ( mean ± SEM ; 3 independent cell lines per genotype ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by ANOVA Single Factor .

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I, J Senescence-associated β-galactosidasestaining (I, blue) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media. (J) Quantitation of the number of senescent cells shown in (I) (mean ± SEM; 3 independent cell lines per genotype).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.


A , B Dot plots of FACS cell cycle analyses of WT and SirT7 - / - MEFs in passage 3 ( P3 ) and 6 ( P6 ) using EdU incorporation and 7AAD ( A ) . Percentages of cells in Sub G1 ( apoptotic cells , left square ) and cells with DNA content above 4N ( polyploid , right square ) . ( B ) Quantitation of experiment shown in ( A ) ( mean ± SEM ; 3 samples per genotype ) .

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A, B Dot plots of FACS cell cycle analyses of WT and SirT7-/- MEFs in passage 3 (P3) and 6 (P6) using EdU incorporation and 7AAD (A). Percentages of cells in Sub G1 (apoptotic cells, left square) and cells with DNA content above 4N (polyploid, right square). (B) Quantitation of experiment shown in (A) (mean ± SEM; 3 samples per genotype).


C Survival curve for WT and SirT7 - / - thymocytes after X - Ray irradiation ( IR ) at the indicated doses . Cell death was quantified by FACS using AnexinV and 7AAD staining 18 hours post insult ( mean ± SEM ; 3 samples per genotype from one of two independent experiments ) .

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C Survival curve for WT and SirT7-/- thymocytes after X-Ray irradiation (IR) at the indicated doses. Cell death was quantified by FACS using AnexinV and 7AAD staining 18 hours post insult (mean ± SEM; 3 samples per genotype from one of two independent experiments).


D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X - Ray doses , then plated at low density . After 9 days , colonies were stained with crystal violet and counted ( mean ± SEM ; 3 independent cell lines per genotype ) .

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D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X-Ray doses, then plated at low density. After 9 days, colonies were stained with crystal violet and counted (mean ± SEM; 3 independent cell lines per genotype).


F Quantitation of experiment shown in ( E ) ( mean ± SEM ; 9 WT and 3 SirT7 - / - samples ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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F Quantitation of experiment shown in (E) (mean ± SEM; 9 WT and 3 SirT7-/- samples).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


A Quantitation of neutral comet assays , using passage 3 WT and SirT7 - / - primary MEFs , showing ( left ) the amount ( % ) of DNA in the tail and ( right ) the tail moment ( see Fig S3A for representative images ) .

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A Quantitation of neutral comet assays, using passage 3 WT and SirT7-/- primary MEFs, showing (left) the amount (%) of DNA in the tail and (right) the tail moment (see Fig S3A for representative images).


B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7 - / - primary fibroblasts after IR ( 8Gy ) . ATM inhibitor ( ATMi ) KU - 55933 was added 30 min prior to irradiation where indicated . One representative blot from 4 independent experiments is shown .

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B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7-/- primary fibroblasts after IR (8Gy). ATM inhibitor (ATMi) KU-55933 was added 30 min prior to irradiation where indicated. One representative blot from 4 independent experiments is shown.


C - F IF analysis of WT and SirT7 - / - primary fibroblasts showing γH2AX dynamics after DNA damage induction . Cells were untreated ( NoIR ) or treated with 1Gy of X - rays ( IR ) and fixed at the indicated times post insult . Cells were pulsed with EdU ( green ) 30 min prior to fixation , then stained for γH2AX ( red ) and counterstained with DAPI ( blue ) ( n > 30 cells per group / mice ; 3 mice per genotype ) . ( C ) Representative images of untreated S - phase ( NoIR , EdU positive ) WT and SirT7 - / - nuclei ( scale bar 5µm ) . ( D ) Quantitation of the number of γH2AX foci per nucleus at the indicated time points post IR ( 1Gy ) , and indicated cell cycle phases . ( E ) Quantitation of experiment described in ( C ) showing mean number of γH2AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 ( mean ± SEM from 3 independent experiments ) . Total γH2AX foci and γH2AX foci overlapping with or at the periphery of heterochromatic regions were enumerated . Nuclei and pericentric heterochromatin ( chromocenters ) were segmented by DAPI staining . Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number .

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C-F IF analysis of WT and SirT7-/- primary fibroblasts showing γH2AX dynamics after DNA damage induction. Cells were untreated (NoIR) or treated with 1Gy of X-rays (IR) and fixed at the indicated times post insult. Cells were pulsed with EdU (green) 30 min prior to fixation, then stained for γH2AX (red) and counterstained with DAPI (blue) (n>30 cells per group/mice; 3 mice per genotype). (C) Representative images of untreated S-phase (NoIR, EdU positive) WT and SirT7-/- nuclei (scale bar 5µm). (D) Quantitation of the number of γH2AX foci per nucleus at the indicated time points post IR (1Gy), and indicated cell cycle phases. (E) Quantitation of experiment described in (C) showing mean number of γH2AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 (mean ± SEM from 3 independent experiments). Total γH2AX foci and γH2AX foci overlapping with or at the periphery of heterochromatic regions were enumerated. Nuclei and pericentric heterochromatin (chromocenters) were segmented by DAPI staining. Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number.


( F ) Representative images of WT and SirT7 - / - primary fibroblasts in G2 phase showing γH2AX ( red ) and DAPI ( blue ) at the indicated period of time after IR . ( Right ) 3D rendering of the IF segmentation depicting nuclei ( pale blue ) , chromocenters ( darker blue ) , and γH2AX ( yellow denotes foci associated with pericentric heterochromatin , otherwise foci are red ) . Scale bar 2 µm .

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(F) Representative images of WT and SirT7-/- primary fibroblasts in G2 phase showing γH2AX (red) and DAPI (blue) at the indicated period of time after IR. (Right) 3D rendering of the IF segmentation depicting nuclei (pale blue), chromocenters (darker blue), and γH2AX (yellow denotes foci associated with pericentric heterochromatin, otherwise foci are red). Scale bar 2 µm.


G FACS quantitation of WT and SirT7 - / - MEF cells in S - phase after insult with 10 mM hydroxyurea ( HU ) for 24 hours . Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS ( mean ± SEM ; 5 samples per genotype ) .

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G FACS quantitation of WT and SirT7-/- MEF cells in S-phase after insult with 10 mM hydroxyurea (HU) for 24 hours. Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS (mean ± SEM; 5 samples per genotype).


H , I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7 - / - MEFs . ( H ) Representative images from cells labeled for 20 min with IdU ( green ) followed by 20 min of CldU ( red ) . ( I ) Quantitation of fork velocity ( fiber length / labeling time ; mean ± SEM ; 3 samples per genotype per condition ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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H, I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7-/- MEFs. (H) Representative images from cells labeled for 20 min with IdU (green) followed by 20 min of CldU (red). (I) Quantitation of fork velocity (fiber length/labeling time; mean ± SEM; 3 samples per genotype per condition).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


A - E IF analysis of WT and SirT7 - / - primary fibroblasts after IR ( 1Gy ) and 1 hour chase . Cells were pulsed with EdU 30 min prior to fixation , stained with antibodies against γH2AX and 53BP1 and then counterstained with DAPI ( scale bar 5µm ) . ( A ) Representative images from each cell cycle stage showing merge ( top - left ) including EdU ( green ) , 53BP1 ( top - right , magenta ) , γH2AX ( bottom - left , red ) , and 3D rendering of reconstructed Z - stacks with 53BP1 foci modeled as spheres ( bottom - right ; pale - blue : nucleus ; magenta spheres : 53BP1 foci ) . ( B ) Quantitation of the number of 53BP1 foci per nucleus , and ( C ) mean volume of 53BP1 foci ( n > 30 cells per group / mice ; 3 mice per genotype ) . ( D - E ) Same as in ( B - C ) , upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7 - H188Y in WT and SirT7 - / - primary fibroblasts .

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A-E IF analysis of WT and SirT7-/- primary fibroblasts after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1 and then counterstained with DAPI (scale bar 5µm). (A) Representative images from each cell cycle stage showing merge (top-left) including EdU (green), 53BP1 (top-right, magenta), γH2AX (bottom-left, red), and 3D rendering of reconstructed Z-stacks with 53BP1 foci modeled as spheres (bottom-right; pale-blue: nucleus; magenta spheres: 53BP1 foci). (B) Quantitation of the number of 53BP1 foci per nucleus, and (C) mean volume of 53BP1 foci (n>30 cells per group/mice; 3 mice per genotype). (D-E) Same as in (B-C), upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7-H188Y in WT and SirT7-/- primary fibroblasts.


F Detection of endogenous chromatin - bound and nucleoplasmic 53BP1 protein from WT and SirT7 - / - MEFs before and after IR ( 8Gy ) by western blot . Histone H3 was used as loading control , and tubulin as control for fractionation .

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F Detection of endogenous chromatin-bound and nucleoplasmic 53BP1 protein from WT and SirT7-/- MEFs before and after IR (8Gy) by western blot. Histone H3 was used as loading control, and tubulin as control for fractionation.


G Same as ( F ) , using 293T - REX cells treated with doxycycline or vehicle to induce SIRT7 - HA expression .

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G Same as (F), using 293T-REX cells treated with doxycycline or vehicle to induce SIRT7-HA expression.


H ChIP - on break assay . ( Top ) Schematic of I - SceI substrate construct introduced into HT1080 cells , which contains a single I - SceI site located within a puromycin resistance cassette . Shown along the top are the coordinates for amplicons probed by Q - PCR . ( Bottom ) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells . Q - PCR measurements were normalized to input DNA and non - I - SceI treated samples ( No Cut ) ( mean ± SEM ; one of two independent experiments shown ) . ( Bottom Right ) western blot demonstrating efficient knockdown of SirT7 , with Histone H3 as a loading control .

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H ChIP-on break assay. (Top) Schematic of I-SceI substrate construct introduced into HT1080 cells, which contains a single I-SceI site located within a puromycin resistance cassette. Shown along the top are the coordinates for amplicons probed by Q-PCR. (Bottom) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells. Q-PCR measurements were normalized to input DNA and non-I-SceI treated samples (No Cut) (mean ± SEM; one of two independent experiments shown). (Bottom Right) western blot demonstrating efficient knockdown of SirT7, with Histone H3 as a loading control.


I , J NHEJ repair assay using GFP expression - based reporter system in SirT7 - depleted HT1080 cells ( SirT7 Knockdown ) versus control ( Scramble Control ) . ( I ) Representative FACS dot plots . ( J ) Quantitation of ( I ) ; mean ± SEM of 3 independent clones per condition . Values are normalized to control ( Scramble Control + I - SceI ) mean .

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I, J NHEJ repair assay using GFP expression-based reporter system in SirT7-depleted HT1080 cells (SirT7 Knockdown) versus control (Scramble Control). (I) Representative FACS dot plots. (J) Quantitation of (I); mean ± SEM of 3 independent clones per condition. Values are normalized to control (Scramble Control + I-SceI) mean.


K , L Class - switch recombination in splenic B cells from WT and SirT7 - / - mice stimulated with lipopolysaccharides ( LPS ) and interleukin 4 ( IL4 ) . ( K ) The switching from IgM to IgG1 ( left ) and to IgG3 ( right ) was measured by FACS . ( L ) Quantitation of ( K ) showing mean ± SEM of 3 - 4 samples per genotype . One representative experiment from two is shown . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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K, L Class-switch recombination in splenic B cells from WT and SirT7-/- mice stimulated with lipopolysaccharides (LPS) and interleukin 4 (IL4). (K) The switching from IgM to IgG1 (left) and to IgG3 (right) was measured by FACS. (L) Quantitation of (K) showing mean ± SEM of 3-4 samples per genotype. One representative experiment from two is shown.*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


A , B H3K18Ac IF in WT and SirT7 - / - fibroblasts . Cells were pulsed with EdU ( green ) , stained with antibody against H3K18Ac ( magenta ) and then counterstained with DAPI ( blue ) . ( A ) Representative IF images of late S - phase nuclei ( EdU positive ; scale bar 5µm ) . ( B ) Quantitation of ( A ) throughout the cell cycle .

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A, B H3K18Ac IF in WT and SirT7-/- fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta) and then counterstained with DAPI (blue). (A) Representative IF images of late S-phase nuclei (EdU positive; scale bar 5µm). (B) Quantitation of (A) throughout the cell cycle.


C Same as in ( B ) , in S - phase cells at the indicated times after IR ( 1Gy ) ( mean ± SEM of > 30 cells per cell cycle , time point , and genotype ) .

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C Same as in (B), in S-phase cells at the indicated times after IR (1Gy) (mean ± SEM of >30 cells per cell cycle, time point, and genotype).


D Laser - induced DNA damage in HT1080 cells expressing HA - tagged SirT7 stained with antibodies against γH2AX ( red ) and HA ( green ) , then counterstained with DAPI ( blue ) 30 min post damage ( scale bar 3 µm ) .

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D Laser-induced DNA damage in HT1080 cells expressing HA-tagged SirT7 stained with antibodies against γH2AX (red) and HA (green), then counterstained with DAPI (blue) 30 min post damage (scale bar 3 µm).


E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H ( mean ± SEM ; one of two independent experiments is shown ) .

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E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H (mean ± SEM; one of two independent experiments is shown).


F Recruitment kinetics of GFP - tagged SirT7 after laser - induced microirradiation . ( Top ) Representative cell at the indicated times after induction of DNA damage ( white circle ; scale bar 2µm ) . Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal . ( Middle ) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU - 55933 ATM inhibitor ( ATMi ) , 10M Olaparib PARP inhibitor ( PARPi ) , or DMSO . KU - 55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage ( mean ± SEM ; sample size : SIRT7 - GFP , n = 34 ; SIRT7 - GFP + ATMi , n = 36 ; SIRT7 - GFP + PARPi = 18 ) . ( Bottom ) Same as ( middle ) except quantitation of SIRT7 - GFP relative intensity within the nucleolus . Data was acquired at five second intervals over a span of five min .

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F Recruitment kinetics of GFP-tagged SirT7 after laser-induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2µm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU-55933 ATM inhibitor (ATMi), 10M Olaparib PARP inhibitor (PARPi), or DMSO. KU-55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage (mean ± SEM; sample size: SIRT7-GFP, n=34; SIRT7-GFP + ATMi, n=36; SIRT7-GFP + PARPi=18). (Bottom) Same as (middle) except quantitation of SIRT7-GFP relative intensity within the nucleolus. Data was acquired at five second intervals over a span of five min.


G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H . H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells ( bottom left ) , or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors ( PARPi ) ( bottom right ) ( mean ± SEM ; one of two independent experiments is shown ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by ANOVA Single Factor .

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G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H. H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells (bottom left), or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors (PARPi) (bottom right) (mean ± SEM; one of two independent experiments is shown).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.


A - H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3 - WT , H3 - K18Q or H3 - K18R ( naive , acetylated or deacetylated H3K18 residues , respectively ) . A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette ( mean number of puromycin resistant colonies normalized by plating efficiency ± SEM ; One representative transfection from 3 is shown ) .

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A-H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3-WT, H3-K18Q or H3-K18R (naive, acetylated or deacetylated H3K18 residues, respectively).A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette (mean number of puromycin resistant colonies normalized by plating efficiency ± SEM; One representative transfection from 3 is shown).


B NHEJ repair assay using GFP expression - based reporter system described in Fig 5I ( mean ± SEM of 3 independent experiments . See Fig EV5B for representative FACS dot plots ) .

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B NHEJ repair assay using GFP expression-based reporter system described in Fig 5I (mean ± SEM of 3 independent experiments. See Fig EV5B for representative FACS dot plots).


C - D IF staining of NIH3T3 cells after IR ( 1Gy ) and 1 hour chase . Cells were pulsed with EdU 30 min prior to fixation , stained with antibodies against γH2AX and 53BP1 , and counterstained with DAPI . Representative images can be found in Fig EV5C . ( C ) Quantitation of the number of 53BP1 foci per nucleus , and ( D ) mean volume of 53BP1 foci ( mean ± SEM of > 30 cells per condition and cell cycle stage ) .

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C-D IF staining of NIH3T3 cells after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1, and counterstained with DAPI. Representative images can be found in Fig EV5C. (C) Quantitation of the number of 53BP1 foci per nucleus, and (D) mean volume of 53BP1 foci (mean ± SEM of >30 cells per condition and cell cycle stage).


E Western blot analysis of 53BP1 in chromatin and non - chromatin fractions from NIH3T3 cells expressing H3 - WT , H3 - K18Q or H3 - K18R vectors and exposed to IR ( 10Gy ) . One representative blot is shown from 3 independent experiments . Shown is 53BP1 , GAPDH for fractionation control , and H3 for loading control .

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E Western blot analysis of 53BP1 in chromatin and non-chromatin fractions from NIH3T3 cells expressing H3-WT, H3-K18Q or H3-K18R vectors and exposed to IR (10Gy). One representative blot is shown from 3 independent experiments. Shown is 53BP1, GAPDH for fractionation control, and H3 for loading control.


F - H Same as in ( C - D ) except using antibodies against γH2AX ( red ) and RIF1 ( magenta ) . ( F ) Representative images from cells in S - phase ( EdU positive ; scale bar 5µm ) . ( G ) Quantitation of the number of RIF1 foci per nucleus , and ( H ) mean volume of RIF1 foci ( mean ± SEM of > 30 cells per condition and cell cycle stage ) .

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F-H Same as in (C-D) except using antibodies against γH2AX (red) and RIF1 (magenta). (F) Representative images from cells in S-phase (EdU positive; scale bar 5µm). (G) Quantitation of the number of RIF1 foci per nucleus, and (H) mean volume of RIF1 foci (mean ± SEM of >30 cells per condition and cell cycle stage).


I 53BP1 enrichment at the indicated loci as described in Figure 5H in Scramble control or SirT7 knockdown cells expressing the indicated H3 constructs ( mean ± SEM ; one of two independent experiments ) .

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I 53BP1 enrichment at the indicated loci as described in Figure 5H in Scramble control or SirT7 knockdown cells expressing the indicated H3 constructs (mean ± SEM; one of two independent experiments).


J Western blot analysis of chromatin fractions from primary WT and SirT7 - / - MEFs expressing the indicated H3 constructs and exposed to IR ( 10Gy ) . Shown are levels of 53BP1 , SIRT7 , H3 - Myc , GAPDH for fractionation control , and H3 for loading control .

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J Western blot analysis of chromatin fractions from primary WT and SirT7-/- MEFs expressing the indicated H3 constructs and exposed to IR (10Gy). Shown are levels of 53BP1, SIRT7, H3-Myc, GAPDH for fractionation control, and H3 for loading control.


B : Consequences of ROS on the SUMO proteome vary amongst cell lines . HeLa , MCF7 , U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N - Ethylmaleimide ( NEM ) . Samples were analyzed by immunoblotting with SUMO1 antibodies .

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B: Consequences of ROS on the SUMO proteome vary amongst cell lines. HeLa, MCF7, U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N-Ethylmaleimide (NEM). Samples were analyzed by immunoblotting with SUMO1 antibodies.


C : H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells . HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min . Cells were lysed with 1 % SDS in PBS buffer and 20 mM NEM . Upon immunoprecipitation of the SUMO1 proteome , samples were analyzed by immunoblotting with TRIM28 antibodies .

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C: H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells. HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min. Cells were lysed with 1% SDS in PBS buffer and 20 mM NEM. Upon immunoprecipitation of the SUMO1 proteome, samples were analyzed by immunoblotting with TRIM28 antibodies.


D : Induction of the Uba2 ~ Ubc9 disulfide is a conserved mechanism . HeLa , MCF7 , U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min , lysed in non reducing buffer supplemented with 20 mM NEM , and analyzed by immunoblotting with Uba2 antibodies .

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D: Induction of the Uba2~Ubc9 disulfide is a conserved mechanism. HeLa, MCF7, U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min, lysed in non reducing buffer supplemented with 20 mM NEM, and analyzed by immunoblotting with Uba2 antibodies.


A : Upper panel : Strategy for the identification of oxidation resistant Ubc9 variants . Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations . Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2 . For this purpose , we used a FRET - based in vitro SUMOylation assay with recombinant SUMO E1 , YFP - SUMO and CFP - RanGAPtail and ATP . Lower panel : Ubc9 W103R is H2O2 resistant . Bacterial lysates containing Ubc9 wt ( left panel ) or Ubc9 W103R ( right panel ) were tested as described .

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A: Upper panel: Strategy for the identification of oxidation resistant Ubc9 variants. Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations. Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2. For this purpose, we used a FRET-based in vitro SUMOylation assay with recombinant SUMO E1, YFP-SUMO and CFP-RanGAPtail and ATP. Lower panel: Ubc9 W103R is H2O2 resistant. Bacterial lysates containing Ubc9 wt (left panel) or Ubc9 W103R (right panel) were tested as described.


B : Ubc9 W103 mutants are H2O2 resistant but not fully active . Left panel : Recombinant Ubc9 W103R , W103A and W103F were purified ( embedded panel ) . Resistance against oxidation was tested under conditions of limiting E1 enzyme : 21 nM Aos1 / Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP - SUMO1 and CFP - RanGAPtail . Right panel : To compare specific activities of wt and mutants in the absence of H2O2 , sumoylation assays were carried out using limiting Ubc9 concentration . Reactions contained 35 nM Aos1 / Uba2 , 11 nM Ubc9 , 85 nM each of YFP - SUMO1 and CFP - RanGAPtail and 1 mM ATP .

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B: Ubc9 W103 mutants are H2O2 resistant but not fully active. Left panel: Recombinant Ubc9 W103R, W103A and W103F were purified (embedded panel). Resistance against oxidation was tested under conditions of limiting E1 enzyme: 21 nM Aos1/Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP-SUMO1 and CFP-RanGAPtail. Right panel: To compare specific activities of wt and mutants in the absence of H2O2, sumoylation assays were carried out using limiting Ubc9 concentration. Reactions contained 35 nM Aos1/Uba2, 11 nM Ubc9, 85 nM each of YFP-SUMO1 and CFP-RanGAPtail and 1 mM ATP.


B : In FRET - based assay , Ubc9 D100A is active upon H2O2 treatment . Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig . 2B .

B: In FRET-based assay, Ubc9 D100A is active upon H2O2 treatment. Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig. 2B.


C : Ubc9 wt and D100A are equally active in CFP - RanGAPtail SUMOylation . Assays were with 35 nM Aos1 / Uba2 and 15 nM Ubc9 .

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C: Ubc9 wt and D100A are equally active in CFP-RanGAPtail SUMOylation. Assays were with 35 nM Aos1/Uba2 and 15 nM Ubc9.


D : Ubc9 D100A is fully competent to form a thioester with SUMO . Single turnover reactions were performed using 630 nM E1 , 3 , 3 µM SUMO1 and 600 nM Ubc9 . Reactions were stopped at different time points by addition of non - reducing buffer . .

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D: Ubc9 D100A is fully competent to form a thioester with SUMO. Single turnover reactions were performed using 630 nM E1, 3,3 µM SUMO1 and 600 nM Ubc9. Reactions were stopped at different time points by addition of non-reducing buffer..


E : Ubc9 wt and D100A are equally active in PIAS2b - dependent SUMOylation . Assays were with 300 nM GST - p53 , 170 nM E1 , 120 nM Ubc9 , 5 µM SUMO2 and 49 nM GST -

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E: Ubc9 wt and D100A are equally active in PIAS2b - dependent SUMOylation. Assays were with 300 nM GST-p53, 170 nM E1, 120 nM Ubc9, 5 µM SUMO2 and 49 nM GST-Pias2b


F : Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation . Assays were with 650 nM YFP - SP100 , 110 nM of E1 , 25 Ubc9 , 40 nM RanBP2ΔFG and 3 , 5 µM SUMO1 ( Left panel ) or SUMO2 ( Right panel ) .

F: Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation. Assays were with 650 nM YFP-SP100, 110 nM of E1, 25 Ubc9, 40 nM RanBP2ΔFG and 3,5 µM SUMO1 (Left panel) or SUMO2 (Right panel).


A : Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT . 21 nM Aos1 / Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP - SUMO1 and CFP - RanGAPtail diluted in DTT - free buffer . The final DTT concentration in the reaction was 6 µM . Prior to addition of ATP , extra amount of DTT was added at indicated final concentration .

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A: Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT. 21 nM Aos1/Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP-SUMO1 and CFP-RanGAPtail diluted in DTT-free buffer. The final DTT concentration in the reaction was 6 µM. Prior to addition of ATP, extra amount of DTT was added at indicated final concentration.


B : Both Uba2 ~ Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A . 100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2 . One hour after H2O2 addition , 500 µM reduced glutathione was added . Samples were taken at indicated time points and the Uba2 ~ Ubc9 disulfide was monitored by immunoblotting against Uba2 . Lower panel : Quantification of the ratio between crosslinked Uba2 vs total

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B: Both Uba2~Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A.100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2. One hour after H2O2 addition, 500 µM reduced glutathione was added. Samples were taken at indicated time points and the Uba2~Ubc9 disulfide was monitored by immunoblotting against Uba2. Lower panel: Quantification of the ratio between crosslinked Uba2 vs total Uba2


A : Generation of polyclonal populations of stable cells . Top panel : Replacement strategy : U2OS cells transfected with pIRES - hrGFP II - based vectors were selected via addition of G418 . After 4 weeks , low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks . Bottom panel : Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9 : U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected . Cells were lysed in Laemmli buffer and Ubc9 and a - tubulin as loading control were detected by immunoblotting .

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A: Generation of polyclonal populations of stable cells. Top panel: Replacement strategy: U2OS cells transfected with pIRES-hrGFP II-based vectors were selected via addition of G418. After 4 weeks, low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks. Bottom panel: Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9: U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected. Cells were lysed in Laemmli buffer and Ubc9 and a-tubulin as loading control were detected by immunoblotting.


B : Ubc9 D100A is severely impaired for disulfide bond formation with Uba2 . Stable U2OS cells were transfected with siRNA against Ubc9 for 72h , treated with 250 µM H2O2 for different time points , from 2 min to 2 hours , lysed in non - reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide ( upper panel ) or free Uba2 as loading control ( see also Appendix Fig . S5 ) . Lower panel : relative intensities of Ubc9 bands were measured using imageJ . Highest intensities in both cell population were set to 1 .

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B: Ubc9 D100A is severely impaired for disulfide bond formation with Uba2. Stable U2OS cells were transfected with siRNA against Ubc9 for 72h, treated with 250 µM H2O2 for different time points, from 2 min to 2 hours, lysed in non-reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide (upper panel) or free Uba2 as loading control (see also Appendix Fig. S5). Lower panel: relative intensities of Ubc9 bands were measured using imageJ. Highest intensities in both cell population were set to 1.


C : Ubc9 D100A impairs cell survival upon H2O2 treatment . Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h , transferred into 6 well plates ( 20 000 cells / well ) and treated with increasing H2O2 concentration for 1h . Ten days later , cells were washed with PBS , fixed with 4 % formaldehyde and stained with crystal violet . The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin . An arbitrary value of 1 was given to the area occupied by non - treated cells . The graph is showing the mean of 4 independent experiments . Error bars are S . E . M . * : p < 0 .

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C: Ubc9 D100A impairs cell survival upon H2O2 treatment. Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h, transferred into 6 well plates (20 000 cells / well) and treated with increasing H2O2 concentration for 1h. Ten days later, cells were washed with PBS, fixed with 4% formaldehyde and stained with crystal violet. The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin. An arbitrary value of 1 was given to the area occupied by non-treated cells. The graph is showing the mean of 4 independent experiments. Error bars are S.E.M. *: p < 0.05


D : Constitutive expression of Ubc9 D100A affects cellgrowth . 103 cells were plated per 10 cm plate and cultivated for 10 days in 20 % or 5 % O2 incubators . Subsequently , cells were colored using crystal violet . The area of each clone was measured using ImageJ . Representative plates are shown in the upper panel . Lower left panel : Distribution of colony sizes , shown for one of the three biological replicates . Lower right panel : Comparison of the colonies size between " Ubc9 wt " and " Ubc9 DA " cells . The average size of " Ubc9 wt " was set to 1 . Shown is the mean of 3 independent experiments , each of which was performed in technical triplicates . Error bars are S . E . M .

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D: Constitutive expression of Ubc9 D100A affects cellgrowth. 103 cells were plated per 10 cm plate and cultivated for 10 days in 20% or 5% O2 incubators. Subsequently, cells were colored using crystal violet. The area of each clone was measured using ImageJ. Representative plates are shown in the upper panel. Lower left panel: Distribution of colony sizes, shown for one of the three biological replicates. Lower right panel: Comparison of the colonies size between "Ubc9 wt"and "Ubc9 DA" cells. The average size of "Ubc9 wt" was set to 1. Shown is the mean of 3 independent experiments, each of which was performed in technical triplicates. Error bars are S.E.M.


A : Phosphorylation of ATM is impaired in Ubc9 D100A cells . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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A: Phosphorylation of ATM is impaired in Ubc9 D100A cells. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


B : Phosphorylation of gamma - H2AX is impaired in Ubc9 D100A . Experiment was performed as in A . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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B: Phosphorylation of gamma-H2AX is impaired in Ubc9 D100A. Experiment was performed as in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


C : Phosphorylation of Chk2 is impaired in Ubc9 D100A . Experiment was performed as described in A . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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C: Phosphorylation of Chk2 is impaired in Ubc9 D100A. Experiment was performed as described in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


D : Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure . Stable U2OS cell populations were exposed to 2 . 5 mM hydroxyurea or 250 µM H2O2 . Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies .

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D: Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure. Stable U2OS cell populations were exposed to 2.5 mM hydroxyurea or 250 µM H2O2. Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies.