Reverse Genetic Analysis of Homologous DNA Recombination in Higher Eukaryotes

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Chromosomal double-stand breaks (DSBs) arise after ionizing irradiation and during DNA replication. A chromosomal break is a lethal event, if it left unrepaired. There are two major DSB repair pathways, homologous DNA recombination (HR) and nonhomologous DNA end-joining, which are highly conserved from yeast to mammals. Rad51, which is structurally and functionally related to the Escherichia coli recombination protein RecA, Rad51B, Rad54 and Mre11 are involved in HR, while the Ku proteins are involved in end-joining. HR-deficient. yeast cells are capable of proliferating, though murine cells deficient in Rad51 or Mre11 are not viable. To investigate the essential roles of HR in vertebrate cells, we have generated conditionally Rad51-and Mre11-deficient cells from hyper-recombinogenic chicken B lymphocyte line DT40. The depletion of Rad51 or Mre11 caused the appearance of chromosomal breaks and subsequent cell death. These observations indicate that HR is required to repair spontaneousl … More y-arising DSBs, possibly during DNA replication. To further analyze the involvement of HR in repairing spontaneous DNA lesions, we measured the level of sister chromatid exchange (SCE) in HR-deficient cells, which is induced by crosslinking agents and known to be intimately associated with DNA replication. The levels of both spontaneous and induced SCE were strongly reduced in HR-deficient cells including RAD51ィイD1-/-ィエD1, Rad51BィイD1-/-ィエD1 and RAD54ィイD1-/-ィエD1 cells, but not in KU70ィイD1-/-ィエD1 cells, confirming that HR is indeed the mechanism responsible for SCE. HR may use the nascent sister chromatid to repair potentially lethal DNA lesions accomp anying replication. Since crossing-over is a relatively infrequent event in yeast mitosis, the presence of as many as 1-5 SCEs (i. e., visible cross-overs) implies that recombinational rep air without such crossovers may also occur frequently in the cycling human cells. Conceivably, HR has to play a more important role in maintaining chromosomal DNA in vertebrate cells than it does in yeast, probably due to the several hundred-fold difference in genome size between vertebrates and lower eukaryotes. Less