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XRCC1 interacts with the p58 subunit of DNA Pol alpha-primase and may coordinate DNA repair and replication during S phase.

Lévy N, Oehlmann M, Delalande F, Nasheuer HP, Van Dorsselaer A, Schreiber V, de Murcia G, Ménissier-de Murcia J, Maiorano D, Bresson A - Nucleic Acids Res. (2009)

Bottom Line: In vitro experiments reveal that the binding of poly(ADP-ribose) to p58 inhibits primase activity by competition with its DNA binding property.Addition of recombinant XRCC1-BRCT1 to Xenopus egg extracts slows down DNA synthesis and inhibits the binding of PCNA, but not MCM2 to alkylated chromatin, thus indicating interference with the assembly of functional replication forks.Altogether these results suggest a critical role for XRCC1 in connecting the SSBR machinery with the replication fork to halt DNA synthesis in response to DNA damage.

View Article: PubMed Central - PubMed

Affiliation: FRE 3211, Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, CNRS/Université de Strasbourg, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard S. Brant, BP 10413, F-67412, Illkirch Cedex, France.

ABSTRACT
Repair of single-stranded DNA breaks before DNA replication is critical in maintaining genomic stability; however, how cells deal with these lesions during S phase is not clear. Using combined approaches of proteomics and in vitro and in vivo protein-protein interaction, we identified the p58 subunit of DNA Pol alpha-primase as a new binding partner of XRCC1, a key protein of the single strand break repair (SSBR) complex. In vitro experiments reveal that the binding of poly(ADP-ribose) to p58 inhibits primase activity by competition with its DNA binding property. Overexpression of the XRCC1-BRCT1 domain in HeLa cells induces poly(ADP-ribose) synthesis, PARP-1 and XRCC1-BRCT1 poly(ADP-ribosyl)ation and a strong S phase delay in the presence of DNA damage. Addition of recombinant XRCC1-BRCT1 to Xenopus egg extracts slows down DNA synthesis and inhibits the binding of PCNA, but not MCM2 to alkylated chromatin, thus indicating interference with the assembly of functional replication forks. Altogether these results suggest a critical role for XRCC1 in connecting the SSBR machinery with the replication fork to halt DNA synthesis in response to DNA damage.

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GST-XRCC1-BRCT1 overexpressed in HeLa cells triggers PAR synthesis and is poly(ADPribosyl)ated. HeLa cells overexpressing GST-hXRCC1-BRCT1 (amino acids 282–428, lanes 1–3), GST-hXRCC1-BRCT2 (amino acids 427–633, lanes 4–6) or GST (lanes 7, 8) were either untreated (lanes 1, 4 and 7) or treated with 2.5 mM MMS for 30 min (lanes 2, 5 and 8). Where indicated, cells were pre-incubated for 2 h with 100 nM of the PARP inhibitor Ku-0058948 (lanes 3 and 6). (A) Inputs corresponding to 2% of each lysates. Proteins were analyzed by GST pull down and western blot (B) using successively the anti-PAR, anti-PARP-1 and anti-GST antibodies.
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Figure 4: GST-XRCC1-BRCT1 overexpressed in HeLa cells triggers PAR synthesis and is poly(ADPribosyl)ated. HeLa cells overexpressing GST-hXRCC1-BRCT1 (amino acids 282–428, lanes 1–3), GST-hXRCC1-BRCT2 (amino acids 427–633, lanes 4–6) or GST (lanes 7, 8) were either untreated (lanes 1, 4 and 7) or treated with 2.5 mM MMS for 30 min (lanes 2, 5 and 8). Where indicated, cells were pre-incubated for 2 h with 100 nM of the PARP inhibitor Ku-0058948 (lanes 3 and 6). (A) Inputs corresponding to 2% of each lysates. Proteins were analyzed by GST pull down and western blot (B) using successively the anti-PAR, anti-PARP-1 and anti-GST antibodies.

Mentions: The fact that PAR affects primase activity and hXRCC1-BRCT1 interacts with p58 prompted us to examine whether hXRCC1-BRCT1 could be poly(ADP-ribosyl)ated in vivo. GST-tagged hXRCC1-BRCT1 overexpressed in undamaged HeLa cells was slightly poly(ADP-ribosyl)ated, in contrast to GST alone or GST-hXRCC1-BRCT2 which were not poly(ADP-rybosyl)ated (Figure 4B, compare lane 1 with lanes 4 and 7). Treatment of cells with 2.5 mM MMS for 30 min triggered PARP-1 activation and PAR synthesis, and cells overexpressing GST-hXRCC1-BRCT1 displayed high levels of automodified PARP-1 suggesting that hXRCC1-BRCT1 overexpression stimulates PARP-1 activity (Figure 4B, lanes 2, 5 and 8). In addition, GST-hXRCC1-BRCT1 was poly(ADP-ribosyl)ated in MMS-treated cells and this modification decreased in the presence of the PARP inhibitor Ku-0058948 (Figure 4B, lanes 2 and 3). The signals recognized by the PAR antibody with a molecular weight higher than 116 kDa, co-purifying with GST-hXRCC1-BRCT1 correspond to automodified PARP-1 which strongly interacts with the BRCT1 domain of XRCC1 (5). The observation that GST-hXRCC1-BRCT1 is poly(ADP-ribosyl)ated in vivo confirmed previous in vitro data showing that this domain could be covalently poly(ADP-ribosyl)ated by PARP-1 (and PARP-2) in addition to its non-covalent binding to PAR (6). The increased PAR level observed in undamaged GST-hXRCC1-BRCT1 expressing cells was confirmed by immunofluorescence microscopy. PAR synthesis was detected in HeLa cells overexpressing GFP-hXRCC1-BRCT1 but not GFP-hXRCC1-BRCT2 (Supplementary Figure 1). Treatment of cells with hydrogen peroxide triggered higher amount of PAR produced in GFP-hXRCC1-BRCT1 expressing cells compared to untransfected or GFP-hXRCC1-BRCT2 expressing cells (Supplementary Figure 1). In addition, some PAR foci colocalized with GFP-hBRCT1 but not with GFP-hBRCT2.Figure 4.


XRCC1 interacts with the p58 subunit of DNA Pol alpha-primase and may coordinate DNA repair and replication during S phase.

Lévy N, Oehlmann M, Delalande F, Nasheuer HP, Van Dorsselaer A, Schreiber V, de Murcia G, Ménissier-de Murcia J, Maiorano D, Bresson A - Nucleic Acids Res. (2009)

GST-XRCC1-BRCT1 overexpressed in HeLa cells triggers PAR synthesis and is poly(ADPribosyl)ated. HeLa cells overexpressing GST-hXRCC1-BRCT1 (amino acids 282–428, lanes 1–3), GST-hXRCC1-BRCT2 (amino acids 427–633, lanes 4–6) or GST (lanes 7, 8) were either untreated (lanes 1, 4 and 7) or treated with 2.5 mM MMS for 30 min (lanes 2, 5 and 8). Where indicated, cells were pre-incubated for 2 h with 100 nM of the PARP inhibitor Ku-0058948 (lanes 3 and 6). (A) Inputs corresponding to 2% of each lysates. Proteins were analyzed by GST pull down and western blot (B) using successively the anti-PAR, anti-PARP-1 and anti-GST antibodies.
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Figure 4: GST-XRCC1-BRCT1 overexpressed in HeLa cells triggers PAR synthesis and is poly(ADPribosyl)ated. HeLa cells overexpressing GST-hXRCC1-BRCT1 (amino acids 282–428, lanes 1–3), GST-hXRCC1-BRCT2 (amino acids 427–633, lanes 4–6) or GST (lanes 7, 8) were either untreated (lanes 1, 4 and 7) or treated with 2.5 mM MMS for 30 min (lanes 2, 5 and 8). Where indicated, cells were pre-incubated for 2 h with 100 nM of the PARP inhibitor Ku-0058948 (lanes 3 and 6). (A) Inputs corresponding to 2% of each lysates. Proteins were analyzed by GST pull down and western blot (B) using successively the anti-PAR, anti-PARP-1 and anti-GST antibodies.
Mentions: The fact that PAR affects primase activity and hXRCC1-BRCT1 interacts with p58 prompted us to examine whether hXRCC1-BRCT1 could be poly(ADP-ribosyl)ated in vivo. GST-tagged hXRCC1-BRCT1 overexpressed in undamaged HeLa cells was slightly poly(ADP-ribosyl)ated, in contrast to GST alone or GST-hXRCC1-BRCT2 which were not poly(ADP-rybosyl)ated (Figure 4B, compare lane 1 with lanes 4 and 7). Treatment of cells with 2.5 mM MMS for 30 min triggered PARP-1 activation and PAR synthesis, and cells overexpressing GST-hXRCC1-BRCT1 displayed high levels of automodified PARP-1 suggesting that hXRCC1-BRCT1 overexpression stimulates PARP-1 activity (Figure 4B, lanes 2, 5 and 8). In addition, GST-hXRCC1-BRCT1 was poly(ADP-ribosyl)ated in MMS-treated cells and this modification decreased in the presence of the PARP inhibitor Ku-0058948 (Figure 4B, lanes 2 and 3). The signals recognized by the PAR antibody with a molecular weight higher than 116 kDa, co-purifying with GST-hXRCC1-BRCT1 correspond to automodified PARP-1 which strongly interacts with the BRCT1 domain of XRCC1 (5). The observation that GST-hXRCC1-BRCT1 is poly(ADP-ribosyl)ated in vivo confirmed previous in vitro data showing that this domain could be covalently poly(ADP-ribosyl)ated by PARP-1 (and PARP-2) in addition to its non-covalent binding to PAR (6). The increased PAR level observed in undamaged GST-hXRCC1-BRCT1 expressing cells was confirmed by immunofluorescence microscopy. PAR synthesis was detected in HeLa cells overexpressing GFP-hXRCC1-BRCT1 but not GFP-hXRCC1-BRCT2 (Supplementary Figure 1). Treatment of cells with hydrogen peroxide triggered higher amount of PAR produced in GFP-hXRCC1-BRCT1 expressing cells compared to untransfected or GFP-hXRCC1-BRCT2 expressing cells (Supplementary Figure 1). In addition, some PAR foci colocalized with GFP-hBRCT1 but not with GFP-hBRCT2.Figure 4.

Bottom Line: In vitro experiments reveal that the binding of poly(ADP-ribose) to p58 inhibits primase activity by competition with its DNA binding property.Addition of recombinant XRCC1-BRCT1 to Xenopus egg extracts slows down DNA synthesis and inhibits the binding of PCNA, but not MCM2 to alkylated chromatin, thus indicating interference with the assembly of functional replication forks.Altogether these results suggest a critical role for XRCC1 in connecting the SSBR machinery with the replication fork to halt DNA synthesis in response to DNA damage.

View Article: PubMed Central - PubMed

Affiliation: FRE 3211, Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, CNRS/Université de Strasbourg, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard S. Brant, BP 10413, F-67412, Illkirch Cedex, France.

ABSTRACT
Repair of single-stranded DNA breaks before DNA replication is critical in maintaining genomic stability; however, how cells deal with these lesions during S phase is not clear. Using combined approaches of proteomics and in vitro and in vivo protein-protein interaction, we identified the p58 subunit of DNA Pol alpha-primase as a new binding partner of XRCC1, a key protein of the single strand break repair (SSBR) complex. In vitro experiments reveal that the binding of poly(ADP-ribose) to p58 inhibits primase activity by competition with its DNA binding property. Overexpression of the XRCC1-BRCT1 domain in HeLa cells induces poly(ADP-ribose) synthesis, PARP-1 and XRCC1-BRCT1 poly(ADP-ribosyl)ation and a strong S phase delay in the presence of DNA damage. Addition of recombinant XRCC1-BRCT1 to Xenopus egg extracts slows down DNA synthesis and inhibits the binding of PCNA, but not MCM2 to alkylated chromatin, thus indicating interference with the assembly of functional replication forks. Altogether these results suggest a critical role for XRCC1 in connecting the SSBR machinery with the replication fork to halt DNA synthesis in response to DNA damage.

Show MeSH
Related in: MedlinePlus