Cupello S , Lin Y , Yan S .

R01 CA225637 NCI NIH HHS , R15 GM101571 NIGMS NIH HHS , R15 GM114713 NIGMS NIH HHS

             response to oxidative stress
             DNA damage response, detection of DNA damage
             detection of oxidative stress

	Figure 1. XRCC1 is not required for ATR-Chk1 DNA damage response pathway in Xenopus LSS system. (A) Hydrogen peroxide (final concentration 100 mM) was added to mock- or XRCC-depleted LSS, which was supplemented with sperm chromatin and incubated for 45 minutes. Extracts were examined via immunoblotting analysis for Chk1 phosphorylation (i.e., Chk1 P-Ser344) and total Chk1. (B) Quantification of Chk1 P-S344 (normalized to total Chk1) from Panel (A). a.u., arbitrary units. (C) Chromatin fractions from Experiments in Panel (A) were isolated and examined via immunoblotting as indicated. Histone 3 serves as loading control. (D) ATR inhibitor VE-822 (final concentration 10 M) or ATM inhibitor KU55933 (final concentration 100 M) was added to XRCC1-depleted LSS, then supplemented with hydrogen peroxide (final concentration 100 mM) and sperm chromatin. After a 45-minute incubation, total egg extracts were examined via immunoblotting as indicated. (E) Quantification of Chk1 P-S344 (normalized to total Chk1) from Panel (D). a.u., arbitrary units. (F) Hydrogen peroxide (final concentration 100 mM) was added to mock- or XRCC-depleted HSS supplemented with sperm chromatin, followed by a 45-minute incubation. Extracts were examined via immunoblotting analysis for Chk1 and Chk1 P-Ser344. (G) Quantification of Chk1 P-S344 (normalized to total Chk1) from Panel (F). a.u., arbitrary units. (H) Chromatin fractions from Experiments in Panel (F) were isolated and examined via immunoblotting as indicated. (A,C,D,F,H) shows representative results from two independent experiments.
	Figure 2. XRCC1 is dispensable for the repair of SSB or gapped plasmids in Xenopus HSS system. (A) SSB plasmid with a 5’-OH was incubated in mock- or XRCC1-depleted HSS. After different timepoints (0, 5, 30, 90 min), DNA repair products were isolated and examined on agarose gel. * indicates partially contaminated DSB in our prep. (B) DNA repair capacity (%, i.e., intensity of DNA repair products / intensity of DNA repair products and SSB plasmid) from Panel (A) was analyzed using Image J. “n.s.” represents no significance (p>0.05, n=4). (C) SSB plasmid with a 5’-P was added to mock- or XRCC1-depleted HSS. After different times, DNA repair products were isolated and examined on agarose gel. (D) DNA repair capacity (%, i.e., intensity of DNA repair products / intensity of DNA repair products and SSB plasmid) from Panel (C) was analyzed using Image J. “n.s.” represents no significance (p>0.05, n=3). (E) Gapped plasmid with a 5’-OH was added to mock- or XRCC1-depleted HSS. After different times, DNA repair products were isolated and examined on agarose gel. (F) DNA repair capacity (%, i.e., intensity of DNA repair products / intensity of DNA repair products and gapped plasmid) from Panel (E) was analyzed using Image J. “n.s.” represents no significance (p>0.05, n=3). (G) Gapped plasmid with a 5’-P was added to mock- or XRCC1-depleted HSS. After different times, DNA repair products were isolated and examined on agarose gel. (H) DNA repair capacity (%, i.e., intensity of DNA repair products / intensity of DNA repair products and gapped plasmid) from Panel (G) was analyzed using Image J. “n.s.” represents no significance (p>0.05, n=3).
	Figure 3. APE2, but not Pol beta nor Pol alpha, is important for SSB repair in Xenopus HSS system. (A) Recombinant Myc-APE2 was added back to APE2-depleted HSS. Then, SSB plasmid with a 5’-OH was added to mock- or APE2-depleted HSS. After different timepoints (1, 5, 30 min), DNA repair products were isolated and examined on agarose gel. (B) DNA repair capacity (%, i.e., intensity of DNA repair products / intensity of DNA repair products and SSB plasmid) from Panel (A) was analyzed using Image J. ** indicates p<0.01; * indicates p<0.05 (n=3). (C) SSB plasmid with a 5’-OH was incubated in mock- or Pol beta-depleted HSS. After different timepoints (0, 5, 30 min), DNA repair products were isolated and examined on agarose gel. (D) DNA repair capacity (%, i.e., intensity of DNA repair products / intensity of DNA repair products and SSB plasmid) from Panel (C) was analyzed using Image J. “n.s.” represents no significance (p>0.05, n=3). (E) SSB plasmid with a 5’-OH was incubated in the presence of DMSO or Aphidicolin (295 M) in HSS. After different timepoints (0, 5, 30 min), DNA repair products were isolated and examined on agarose gel. (F) DNA repair capacity (%, i.e., intensity of DNA repair products / intensity of DNA repair products and SSB plasmid) from Panel (E) was analyzed using Image J. ** indicates p<0.01; * indicates p<0.05 (n=3). (G) SSB plasmid with a 5’-OH was incubated in mock- or Pol alpha-depleted HSS. After different timepoints (0, 5, 30 min), DNA repair products were isolated and examined on agarose gel. (H) DNA repair capacity (%, i.e., intensity of DNA repair products / intensity of DNA repair products and SSB plasmid) from Panel (G) was analyzed using Image J. “n.s.” represents no significance (p>0.05, n=3). (A,C,G) * indicates partially contaminated DSB in our prep.
	Figure 4. XRCC1 is important to repair DNA damage following oxidative stress in Xenopus egg extracts. (A) Hydrogen peroxide and sperm chromatin were added to mock- or XRCC1-depleted LSS. After a 30-minute incubation, reaction mixture was further examined with COMET assays under alkaline condition. Representative images are shown. (B) Quantification of DNA damage from four reactions shown in panel (A). *** indicates p<0.0001; ** indicates p<0.001. (C) Hydrogen peroxide and sperm chromatin were added to mock- or XRCC1-depleted LSS. After a 30-minute incubation, reaction mixture was further analyzed using COMET assays under neutral condition. Representative images are shown. (D) Quantification of DNA damage from four reactions shown in panel (C). *** indicates p<0.0001; * indicates p<0.01. “n.s.” shows no significance.
	Supplementary Figure S1. (A) Schematic diagram of Xenopus laevis XRCC1. (B) Amino acid sequence alignment of XRCC1 using the Clustal Omega software. Abbreviations: XL XRCC1 (Xenopus laevis XRCC1), AAH45032.1; HS XRCC1 (Homo sapiens XRCC1), NP_006288.2; MM XRCC1 (Mus musculus XRCC1), NP_033558.3. “*” indicates identical residues; “-” represents gaps in the alignment; “:” indicates highly conserved residues; “.” represents moderately conserved residues.
	Supplementary Figure S2. Validation of purified recombinant proteins and the activation of ATR and ATM DDR pathways in Xenopus LSS system. (A) Verification of purified GST-XRCC1 (1 µg) on SDS-PAGE gel. (B) Verification of purified GST (1 µg) and GST-Pol beta (1 µg) on SDS-PAGE gel. (C) Hydrogen peroxide (final concentration of 100 mM) was added to LSS with the presence or absence of chromatin DNA. After a 45 -min incubation, total extract was examined via immunoblotting analysis as indicated.
	Supplementary Figure S3. Target proteins such as XRCC1, Pol beta, and Pol alpha were examined for depletion efficiency in LSS or HSS system. (A) Mock- or XRCC1-depleted LSS was examined via immunoblotting analysis. (B) Mock- or XRCC1-depleted HSS was examined via immunoblotting analysis. (C) Total egg extracts from experiment in Figure 3A was examined for endogenous APE2 and Myc-APE2 via immunoblotting analysis. (D) Mock- or Pol beta-depleted HSS was examined via immunoblotting analysis. (E) Mock- or Pol alpha-depleted HSS was examined for p70 subunit of Pol alpha via immunoblotting analysis. PCNA was utilized as loading control in all panels.
	Supplementary Figure S4. A working model for distinct roles of XRCC1 in genome integrity. Following oxidative stress, different types of DNA damage, including but not limited to SSBs, DSBs, and AP sites, are generated. (Left) SSB: XRCC1 interacts with APE2 but plays very minimal role for APE2’s 3’-5’ exonuclease activity. XRCC1 is dispensable for ATR-Chk1 DDR following oxidative stress. XRCC1 is not required for SSB repair. (Middle) DSB: Oxidative stress-induced DSBs may be repair by HR, NHEJ, and MMEJ. XRCC1 is important for repairing oxidative stress-derived DSBs. (Right) AP site: XRCC1 is important for repairing oxidative stress-induced AP sites, likely in the final step of ligation.

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