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J Cell Sci
2013 Oct 01;126Pt 19:4414-23. doi: 10.1242/jcs.128272.
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Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose).
Wei L
,
Nakajima S
,
Hsieh CL
,
Kanno S
,
Masutani M
,
Levine AS
,
Yasui A
,
Lan L
.
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Single-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. How XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 because (1) phosphorylation sites mutated in XRCC1 (X1 pm) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and (2) phosphorylation of XRCC1 is required for efficient polyubiquitylation of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant upregulation of ubiquitylation, indicating that pARylation of XRCC1 prevents the poly-ubiquitylation. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitylation in live cells.
Fig. 1. The BRCT II domain and phosphorylation of XRCC1 are required for proper kinetics of XRCC1 at sites of damage. (A) Top: scheme of constructs for X1w/oBII and X1 pm used in the experiments. Bottom: response of wild-type XRCC1 to treatment with 4 µM PARP inhibitor PJ34 or 100 µM PARG inhibitor tannic acid, and the response of X1w/oBII and X1 pm to 40 µg/ml MMS-induced damage in HeLa cells at the indicated times. (B) Quantification of HeLa cell fractions with MMS-induced foci before, 10 minutes, and 60 minutes after MMS treatment. (C) Scheme of SSBs induced in XPA-UVDE cells after local UVC irradiation, and damage response of XRCC1 and mutant XRCC1 to XPA-UVDE-induced SSBs after 20 J/m2 UVC irradiation at the indicated time points. (D) Staining of pAR in X1 pm-expressing XPA-UVDE cells 5 minutes and 30 minutes after 20 J/m2 UVC irradiation. Yellow arrows indicate the foci formed by XRCC1 and its mutants.
Fig. 2. The BRCT II domain and phosphorylation of XRCC1 are required for recruitment of repair proteins. (A) Time-dependent accumulation of GFP-tagged full-length and mutant XRCC1 at UVA-laser-induced SSBs in HeLa cells after 405 nm laser irradiation for 10 mseconds. (B) Quantification of the intensity of foci of full-length and mutant XRCC1 in HeLa cells. (C) Accumulation of GFP–Polβ, GFP–LigIIIα, YFP–PNK and GFP–APTX in Xrcc1+/+ and Xrcc1−/− MEF cells after 405 nm laser irradiation for 10 mseconds. There is no accumulation of each protein at SSBs in Xrcc1−/− MEF cells. (D) The damage response of GFP–Polβ and GFP–LigIIIα, but not YFP–PNK and GFP–APTX, at sites of SSBs in Xrcc1−/− MEF cells expressing DsRed–X1 pm after laser irradiation. (E) Damage response of GFP–Polβ, GFP–APTX, and YFP–APTX, but not LigIIIα, at SSBs in Xrcc1−/− MEF cells expressing DsRed–XRCC1w/o BII. Yellow arrowheads show the foci formed by each indicated protein.
Fig. 3. The BRCT II domain binds nicked DNA in vitro. (A) Schematic of domains and deletion mutants of LigIIIα (left) and accumulation of the deletion mutants at SSBs (right) in HeLa cells after laser irradiation. (B) Accumulation kinetics of full-length and deletion mutants of LigIIIα. (C) Purification of the XRCC1 BRCT II domain. (D) Gel shift assay with 0.5 pmol of one nucleotide gap DNA after binding with XRCC1 BRCT II domains at the indicated amounts. (E) GFP-X1w/oBRCTII or GFP-BRCTII were co-transfected with Myc-ubiquitin into HEK293 cells; the cell extracts were pulled down by anti-GFP and detected by anti-myc antibody.
Fig. 4. PARG is necessary for translocation of XRCC1 and LigIIIα from pAR to SSBs. (A) Accumulation and dissociation of XRCC1 and LigIIIα in siPARG- or PARG-inhibitor (100 µM tannic acid)-treated HeLa cells at the indicated time points after laser irradiation. Effects of siPARG in HeLa cells are shown in the right panel. (B) Quantification of the intensity of XRCC1 at sites of laser-induced DNA damage in siPARG- or PARG-inhibitor-treated HeLa cells 3 minutes and 1 hour after laser irradiation. (C) U2OS cells were treated with or without MMS and collected at the indicated times. Collected cells were used for the comet assay to analyze the remaining damage. The tail moments of 100 cells at 5 minutes, 30 minutes, 1 hour and 2 hours after treatment or without treatment were measured. (D) Accumulation and dissociation of X1w/oBII in HeLa cells 3 minutes and 30 minutes after treatment with 100 µM tannic acid PARG inhibitor. (E) Accumulation and dissociation of X1w/oBII in Parg+/+ (squares) and Parg−/− (diamonds) cells at the indicated time points after laser irradiation. Arrows indicate the foci formed by the indicated proteins.
Fig. 5. Polyubiquitylation is regulated by polyADP-ribosylation and phosphorylation of XRCC1. (A) Expression of XRCC1 or X1 pm in U2OS cells expressing GFP-tagged XRCC1 or X1 pm treated with MG132 for 30 minutes. β-actin was used as a loading control. (B) HEK 293 cells co-expressing myc–ubiquitin and GFP-tagged wild-type XRCC1, X1 pm or XRCC1 LL360/361DD were pulled down by anti-GFP and detected by anti-myc antibody.
Fig. 6. A model of XRCC1-mediated repair of the SSB machinery. The repair of SSBs within the cell proceeds with modifications of XRCC1. XRCC1 accumulates at pAR through its BRCT I domain, but will be retained at sites of SSBs through its BRCT II domain after pAR is degraded. Meanwhile, X1 pm shows an impaired ubiquitylation, indicating that phosphorylated XRCC1 is required for efficient ubiquitylation. Finally, XRCC1 might be ubiquitylated and degraded by proteasomes for protein recycling after repair completion. Thus, the repair of SSBs within the cell proceeds with formation and degradation of pAR in a manner that is dependent on other modifications and domains of XRCC1.
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