Interaction of human Rad51 recombination protein with single-stranded DNA binding protein, RPA

Golub, Efim I.; Gupta, Ravindra C.; Haaf, Thomas; Wold, Marc S.; Radding, Charles M.

doi:10.1093/nar/26.23.5388

Cited by 180 publications

(148 citation statements)

References 55 publications

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“…The observation that yeast sgs1 or srs2 mutants die for the accumulation of hyperrecombination events could be extended in human cells according to our findings. Moreover, because it has been recently reported that WRN relocalizes to sites of replication arrest after HU treatment, colocalizing with RPA (Costantinou et al, 2000), and on the basis of the documented interaction of RAD51 with RPA (Golub et al, 1998), our observation could also reinforce the hypothesized interaction of WRN and RAD51 in resolving replication conflicts. …”

supporting

confidence: 80%

Werner's Syndrome Protein Is Required for Correct Recovery after Replication Arrest and DNA Damage Induced in S-Phase of Cell Cycle

Pichierri

Franchitto

Mosesso

et al. 2001

MBoC

136

140

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Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse. INTRODUCTIONWerner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging (Salk et al., 1985) and early onset of various neoplasms, including different types of carcinomas and sarcomas (Goto et al., 1981;Hrabko et al., 1982;Sato et al., 1988). This disorder arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. One of the hallmarks of WS patients is the genomic instability as evidenced by the spontaneous chromosome anomalies and large deletions in many genes (Salk et al., 1985;Fukuchi et al., 1989). It has been demonstrated that WRN exhibits DNA unwinding activity (Gray et al., 1997;Suzuki et al., 1997) and exonuclease activity residing in the N-terminal region (Huang et al., 1998). The cellular function of WRN is still unclear. It has been proposed that helicases are required for various DNA metabolic activities, including progression of replication fork, segregation of newly replicated chromosomes, disruption of the nucleosome structure, DNA supercoiling, transcription, recombination, and repair (Duguet, 1997). In addition, it has been suggested that RecQ family of DNA helicases has an important role in the maintenance of genomic integrity during DNA replication . Studies from yeast show that DNA replication does not proceed normally in absence of RecQ helicase function (Stewart et al., 1997) and in Xenopus laevis the ortholog of WRN is absolutely required for proper formation of replication foci (Yan et al., 1998). Functional interaction between proteins involved in DNA replication, such as replication protein A (RPA) and DNA polymerase ␦, and WRN also have been reported (Shen et al., 1998;Brosh et al., 1999;Kamath-Loeb et al., 2000). Furthermore, in yeast, the RecQlike proteins seem involved in ...

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supporting

confidence: 80%

Werner's Syndrome Protein Is Required for Correct Recovery after Replication Arrest and DNA Damage Induced in S-Phase of Cell Cycle

Pichierri

Franchitto

Mosesso

et al. 2001

MBoC

136

140

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“…RAD51 foci are seen at high levels in cells exposed to DNAdamaging treatments such as ionizing radiation (Haaf et al, 1995;Scully et al, 1997a). Similar foci have also been observed in mitotic S-phase cells and are thought to identify sites where stalled or broken replication forks undergo repair (Tashiro et al, 1996;Golub et al, 1998;Raderschall et al, 1999). Consistent with a role in the repair of DSBs, RAD51 foci form during meiosis at the time when genetically programmed DSBs are generated (Plug et al, 1996;Barlow et al, 1997;Moens et al, 1997;Scully et al, 1997b).…”

Section: Introductionmentioning

confidence: 81%

BRCA2-dependent and independent formation of RAD51 nuclear foci

2003

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The formation of RAD51 foci in response to ionizing radiation (IR) represents an important step in the repair of DNA double-strand breaks. RAD51 foci also appear during S phase and are thought to be required for the restart of stalled or broken replication forks. The RAD51 recombinase interacts directly with the breast cancerassociated tumour suppressor BRCA2, an interaction that is required for normal recombination proficiency, radiation resistance and genome stability. In CAPAN-1 cells, which express a truncated form of BRCA2 that is cytoplasmic because of loss of the nuclear localization signal, the formation of IR-induced RAD51 foci is impaired. In this work, we show that S-phase RAD51 foci form normally in CAPAN-1 cells expressing truncated BRCA2. Moreover, we find that RAD51 specifically associates with chromatin at S phase in a reaction that is BRCA2-independent. The observed BRCA2-dependent and independent formation of RAD51 foci shows that intact BRCA2 is not required for RAD51 focus formation per se, leading us to suggest that S phase and IR-induced RAD51 foci assemble by distinct pathways with defined protein requirements.

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“…Immunostaining techniques can detect assembly of recombination proteins into subnuclear staining foci (23)(24)(25)(26)(27)(28)(29)(30)(31)(32). Several lines of evidence suggest that these foci represent protein oligomers assembled at sites of DSBs and possibly daughter strand gaps as well.…”

Section: Reca-like Recombinases Assemble On Single-strand Dna (Ssdna)mentioning

confidence: 99%

“…Double staining shows that multiple proteins required for recombination assemble at the same sites during recombination. Such experiments in yeast and mammalian cells showed that RPA colocalizes with Rad51 (29,(31)(32)(33). An important question raised by the biochemical interaction of ssb and recombinase is: Does ssb assemble before or after recombinase in vivo?…”

Section: Reca-like Recombinases Assemble On Single-strand Dna (Ssdna)mentioning

confidence: 99%

Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Gasior

Olivares

Ear

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

158

125

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Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55͞57 are required, whereas either Rad52 or Rad55͞57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G1, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.H omologous recombination reactions promote repair of DNA ends formed by double-strand breaks (DSBs) and by replication fork collapse. Recombinational repair also allows cells to replicate past DNA lesions that block the progress of DNA polymerase. In phage T4, recombination is critical for initiating replication. In eukaryotes, homologous recombination is critical for accurate reductional segregation of chromosomes during meiosis. Finally, in prokaryotes, recombination allows horizontal transfer of alleles among and between bacteria and phage.At the center of homologous recombination are the recombinases, proteins that promote the formation of heteroduplex DNA. Of particular importance are the recombinases of the RecA family, including RecA in eubacteria, RadA in archea, Rad51 and Dmc1 in eukaryea, and the bacteriophage T4 UvsX protein.RecA-Like Recombinases Assemble on Single-Strand DNA (ssDNA).

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Interaction of human Rad51 recombination protein with single-stranded DNA binding protein, RPA

Cited by 180 publications

References 55 publications

Werner's Syndrome Protein Is Required for Correct Recovery after Replication Arrest and DNA Damage Induced in S-Phase of Cell Cycle

Werner's Syndrome Protein Is Required for Correct Recovery after Replication Arrest and DNA Damage Induced in S-Phase of Cell Cycle

BRCA2-dependent and independent formation of RAD51 nuclear foci

Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

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