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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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Interaction between XRCC1 and p58 subunit of Pol α-primase. (A) Identification of proteins interacting with GST-XRCC1-BRCT1 by mass spectrometry. Sypro ruby stained gel after GST pull-down of HeLa cell extracts expressing either GST or GST-hXRCC1-BRCT1 fused proteins. (B) Identification of XRCC1 associated p58 by immunoprecipitation of XRCC1 from extracts of HeLa cells treated or not with aphidicolin (A: 5 μg/ml, 16 h) and with or without HU (4 mM, 4 h). Co-purifying p58 and immunoprecipitated XRCC1 were identified by western blot. Inputs represent 5% of the total proteins used in immunoprecipitation (pi: pre immune serum). (C) GST pull-down of p58 with different GST-fused XRCC1 domains overexpressed in HeLa cells. A schematic drawing of XRCC1 domain structure is shown. (D) Indirect immunofluorescence microscopy showing co-localization of XRCC1 and p58 in HeLa cells treated (+ HU) or not (−HU) with 4 mM HU for 4 h.
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Figure 1: Interaction between XRCC1 and p58 subunit of Pol α-primase. (A) Identification of proteins interacting with GST-XRCC1-BRCT1 by mass spectrometry. Sypro ruby stained gel after GST pull-down of HeLa cell extracts expressing either GST or GST-hXRCC1-BRCT1 fused proteins. (B) Identification of XRCC1 associated p58 by immunoprecipitation of XRCC1 from extracts of HeLa cells treated or not with aphidicolin (A: 5 μg/ml, 16 h) and with or without HU (4 mM, 4 h). Co-purifying p58 and immunoprecipitated XRCC1 were identified by western blot. Inputs represent 5% of the total proteins used in immunoprecipitation (pi: pre immune serum). (C) GST pull-down of p58 with different GST-fused XRCC1 domains overexpressed in HeLa cells. A schematic drawing of XRCC1 domain structure is shown. (D) Indirect immunofluorescence microscopy showing co-localization of XRCC1 and p58 in HeLa cells treated (+ HU) or not (−HU) with 4 mM HU for 4 h.

Mentions: To identify new proteins interacting with the BRCT1 domain of hXRCC1, we used recombinant GST-tagged hXRCC1 fragment (amino acids 282–428) to pull down protein partners from HeLa cell extracts. Co-purified proteins were separated by SDS–PAGE and analyzed by mass spectrometry. The identification of proteins already known to interact with XRCC1 such as PARP-1, PCNA and DNA-PKcs validated our analysis (5,14,18). In addition, we identified the p58 subunit of the DNA Pol α-primase as a novel hXRCC1-BRCT1 interacting protein (Figure 1A).Figure 1.


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)

Interaction between XRCC1 and p58 subunit of Pol α-primase. (A) Identification of proteins interacting with GST-XRCC1-BRCT1 by mass spectrometry. Sypro ruby stained gel after GST pull-down of HeLa cell extracts expressing either GST or GST-hXRCC1-BRCT1 fused proteins. (B) Identification of XRCC1 associated p58 by immunoprecipitation of XRCC1 from extracts of HeLa cells treated or not with aphidicolin (A: 5 μg/ml, 16 h) and with or without HU (4 mM, 4 h). Co-purifying p58 and immunoprecipitated XRCC1 were identified by western blot. Inputs represent 5% of the total proteins used in immunoprecipitation (pi: pre immune serum). (C) GST pull-down of p58 with different GST-fused XRCC1 domains overexpressed in HeLa cells. A schematic drawing of XRCC1 domain structure is shown. (D) Indirect immunofluorescence microscopy showing co-localization of XRCC1 and p58 in HeLa cells treated (+ HU) or not (−HU) with 4 mM HU for 4 h.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Interaction between XRCC1 and p58 subunit of Pol α-primase. (A) Identification of proteins interacting with GST-XRCC1-BRCT1 by mass spectrometry. Sypro ruby stained gel after GST pull-down of HeLa cell extracts expressing either GST or GST-hXRCC1-BRCT1 fused proteins. (B) Identification of XRCC1 associated p58 by immunoprecipitation of XRCC1 from extracts of HeLa cells treated or not with aphidicolin (A: 5 μg/ml, 16 h) and with or without HU (4 mM, 4 h). Co-purifying p58 and immunoprecipitated XRCC1 were identified by western blot. Inputs represent 5% of the total proteins used in immunoprecipitation (pi: pre immune serum). (C) GST pull-down of p58 with different GST-fused XRCC1 domains overexpressed in HeLa cells. A schematic drawing of XRCC1 domain structure is shown. (D) Indirect immunofluorescence microscopy showing co-localization of XRCC1 and p58 in HeLa cells treated (+ HU) or not (−HU) with 4 mM HU for 4 h.
Mentions: To identify new proteins interacting with the BRCT1 domain of hXRCC1, we used recombinant GST-tagged hXRCC1 fragment (amino acids 282–428) to pull down protein partners from HeLa cell extracts. Co-purified proteins were separated by SDS–PAGE and analyzed by mass spectrometry. The identification of proteins already known to interact with XRCC1 such as PARP-1, PCNA and DNA-PKcs validated our analysis (5,14,18). In addition, we identified the p58 subunit of the DNA Pol α-primase as a novel hXRCC1-BRCT1 interacting protein (Figure 1A).Figure 1.

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