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...
Yeast Rad52 DNA-repair mutants exhibit pronounced radiation sensitivity and a defect in homologous recombination (HR), whereas vertebrate cells lacking Rad52 exhibit a nearly normal phenotype. Biochemical studies show that both yeast Rad52 and Rad55±57 (Rad51 paralogs) stimulate DNA-strand exchange mediated by Rad51. These ®ndings raise the possibility that Rad51 paralogs may compensate for lack of Rad52 in vertebrate cells, explaining the absence of prominent phenotypes for Rad52-de®cient cells. To test this hypothesis, using chicken DT40 cells, we generated conditional mutants de®cient in both RAD52 and XRCC3, which is one of the ®ve vertebrate RAD51 paralogs. Surprisingly, the rad52 xrcc3 doublemutant cells were non-viable and exhibited extensive chromosomal breaks, whereas rad52 and xrcc3 single mutants grew well. Our data reveal an overlapping (but non-reciprocal) role for Rad52 and XRCC3 in repairing DNA double-strand breaks. The present study shows that Rad52 can play an important role in HR repair by partially substituting for a Rad51 paralog. Keywords: DT40/homologous recombination/Rad51 paralogs/Rad52/XRCC3 Introduction DNA double-strand breaks (DSBs) arise during DNA replication and from exposure to agents such as ionizing radiation (IR). A single DSB may cause cell death if left unrepaired. Non-homologous end joining (NHEJ) and homologous recombination (HR) are major DSB repair pathways, and both are conserved across eukaryotic cells (reviewed in Jeggo, 1998;Haber, 1999;Morrison and Takeda, 2000;Sonoda et al., 2001;Thompson and Schild, 2001). The genes involved in HR in the yeast Saccharomyces cerevisiae form the RAD52 epistasis group of genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2) (reviewed in Paques and Haber, 1999). Mutants of these genes are hypersensitive to IR and exhibit mitotic and meiotic recombination defects. Among the members of the Rad52 epistasis group, Rad51, a structural and functional homolog of Escherichia coli RecA, is conserved to the highest degree, exhibiting 69% amino acids sequence identity between S.cerevisiae and humans (Shinohara and Ogawa, 1995). This high degree of conservation suggests that the function of Rad51 is also conserved among eukaryotes. Defective Rad51 is lethal to higher eukaryotic cells, indicating a critical role for HR in repairing spontaneous DSBs arising during DNA replication (Tsuzuki et al., 1996;Sonoda et al., 1998). In vitro studies have shown that yeast and human Rad51 proteins form multimeric helical nucleoprotein ®laments, similar to RecA proteins, which are assembled on single-stranded DNA (ssDNA) or on double-stranded DNA (dsDNA) containing either 5¢ or 3¢ single-stranded tails (Mazin et al., 2000;Sigurdsson et al., 2001). The nucleoprotein ®la-ments mediate the search for homology, strand pairing and strand exchange .Relatives of the Rad51 gene that probably arose by gene duplication and the evolution of new functions (paralogs) are present in yeast and higher eukaryotes ). These Rad51 paralogs include Rad55 and Rad5...
This study evaluated patients for the influence of the dose rate and lung dose of fractionated total body irradiation (TBI) in preparation for allogeneic bone marrow transplantation (BMT) on the subsequent development of interstitial pneumonitis (IP). Sixty-six patients at our institute were treated with TBI followed by BMT. All of the patients received a total TBI dose of 12 Gy given in 6 fractions over 3 days and were divided into 3 groups according to the radiation dose rate and lung dose: group A, lung dose of 8 Gy (n = 18); group B, lung dose of 12 Gy at 8 cGy/min (n = 25); and group C, lung dose of 12 Gy at 19 cGy/min (n = 23). The overall survival rate, the cumulative incidence of relapse, and the cumulative incidence of IP were evaluated in relation to various potential indicators of future IP. There were no significant differences in survival and relapse rates between patient group A and combined groups B and C. Clinically significant IP occurred in 13 patients. The cumulative incidence of IP was significantly higher in patients who developed acute parotitis as indicated by either an elevation in the serum amylase level or parotid pain of grade 1 to 2. There was no difference in IP incidence among groups A, B, and C. There was no significant difference in IP incidence between lung dose values of 8 Gy (with lung shielding) and 12 Gy (without lung shielding) and between dose rate values of 8 cGy/min and 19 cGy/ min, at least when TBI was given in 6 fractions. The presence of acute parotitis during or just after TBI may be a predictor of IP.
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