Chromosome Instability and Defective Recombinational Repair in Knockout Mutants of the Five Rad51 Paralogs

Abstract

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.Double-strand DNA breaks (DSBs) are produced by ionizing radiation (IR) and certain chemicals, and they likely occur frequently during DNA replication (21, 34). A single unrepaired DSB may stimulate cell cycle checkpoints and cause cell death (3, 25). Homologous recombination (HR) has emerged as a major DSB repair pathway in mammalian cells (29,35,44,65,66), as well as in the yeast Saccharomyces cerevisiae. Indeed, the analysis of radiosensitive yeast mutants has revealed a number of key genes involved in HR, which comprise the RAD52 epistasis group (2,32,54), and the HR pathway is conserved from yeast to humans (4,18,53,65). Although yeast is capable of proliferating at a reduced rate in the absence of functional HR, this repair pathway is essential for viability in cycling vertebrate cells for coping with DNA lesions arising during DNA replication (55,56,67,73). This species difference is probably due to the several-hundred-fold difference in genome size between vertebrates and yeast.ScRad51 is closely related to the Escherichia coli recombination protein RecA (5). Among the proteins of the Rad52 epistasis group, Rad51 has the highest degree of structural and functional conservation among all eukaryotes. The high degree of identity of ScRad51 with the human homolog (59% identity) and chicken homolog (59% identity) suggests that Rad51's function is conserved across eukaryotes. A central role for Rad51 in HR in vertebrates is supported by the finding that Rad51 deficiency (36, 55, 67), but not Rad52 or Rad54 deficiency, is lethal to cells (4,18,49,72). In vitro studies show that RecA and Rad51 form multimeric helical nucleoprotein filaments that are assembled on single-stranded DNA (ssDNA) (2). Recent work suggests that the preferred DNA substrat...

Immunoglobulin gene conversion in chicken DT40 cells largely proceeds through an abasic site intermediate generated by excision of the uracil produced by AID‐mediated deoxycytidine deamination

“…It is therefore notable that whilst inhibition of UDG in DT40 does causes a shift IgV gene conversion towards dC/dG point mutation, we do not see a dramatic increase in the overall frequency of point mutation analogous to that which we previously described in DT40 mutants deficient in the RAD51 paralogues XRCC2, XRCC3 or RAD51B [12]. These RAD51 paralogues are likely implicated in homologous-recombinationmediated repair of DNA lesions [13]. One possible explanation for the different phenotypes of DT40 cells deficient in UDG and XRCC2 activity is that many of the AIDgenerated lesions are repaired but by a recombinational pathway involving XRCC2 (and possibly templated on the sister chromatid) but not involving UDG and baseexcision repair.…”

Double-stranded DNA breaks and gene functions in recombination and meiosis

“…The BCDX2 complex binds to nicks of duplex DNA and exhibits ATPase activity in vitro. These results together with the observations that RAD51B [107,108], RAD51C [111], RAD51D [117], and XRCC2 [123] are required for proper formation of RAD51 foci, indicate that these proteins may function as a complex both in assembling the RAD51 complex and in the initial processing of the single-stranded DNA at the damaged sites. Another complex, with RAD51C and XRCC3, was originally detected with a yeast two-hybrid system and then with interaction of proteins expressed in baculovirusinfected insect cells [132][133][134], and then confirmed with human cells lines that express isotope-tagged XRCC3 or RAD51C proteins [135].…”

“…Mutant analysis of RAD51B in a chicken B lymphocyte DT40 cell line indicates that RAD51B is important for maintaining genome integrity and for homologous recombinational repair [107,108]. However, the rad51b mutant cell line is viable; although about 20% cells die after each division as a result of spontaneous chromosomal aberrations, indicating that RAD51B is required for repairing DNA breaks or damages under normal growth conditions.…”

A novel form of ataxia oculomotor apraxia characterized by oxidative stress and apoptosis resistance

“…ATL2ABR cells can be distinguished in their sensitivity to ionizing radiation from A-T cells, which is not surprising as we have previously shown that, with the exception of p53 phosphorylation, the major ATM signalling pathways in response to radiation are intact in these cells. 18 Increased sensitivity to MMC has also been reported in Fanconi's anaemia, 30 in cells with mutations in Mus81, 31 Eme1, 32 Rad51 paralogs 33 and Brca1-deficient cells. 34 The clinical phenotype for this patient shows no resemblance to that for FA, which is characterized by earlyonset aplastic anaemia and cancer susceptibility, 30 neither of which are apparent in this case.…”