Requirement for End-Joining and Checkpoint Functions, but Not RAD52-Mediated Recombination, after EcoRI Endonuclease Cleavage of Saccharomyces cerevisiae DNA

DNA double-strand breaks (DSBs) occur spontaneously, after exposure to ionizing radiation or various chemical clastogens, and as part of normal cellular development. Developmental processes which are initiated by enzymatically induced DSBs include V(D)J recombination in mammalian cells and mating-type switching, intron homing, and meiotic recombination in yeast cells (17, 25, 37, 56, 58, 61). Repair of DSBs in eukaryotic DNA involves complex interactions between proteins associated with chromatin structure, enzymatic repair of broken DNA ends, and cell cycling. In mammalian cells, the primary mechanism of repair of DSBs is through nonhomologous end-joining processes (25, 61, 70). In Saccharomyces cerevisiae, the predominant pathway of DSB repair is through homologous recombination, although other, less common pathways have been described (discussed below). Models for the recombinational repair of DSBs in yeast DNA which involve pairing of broken DNA ends with complementary strands of an unbroken homolog have been proposed (63, 75, 77; see references in reference 56). Recombinational repair of damage-induced DSBs occurring during mitotic growth of yeast cells requires genes within the RAD52 epistasis group, which includes RAD50-59, MRE11, and XRS2 (2, 16, 19, 56, 65). RAD52 group mutants are hypersensitive to ionizing radiation and methyl methanesulfonate but display only a slight sensitivity to UV light. Physical analysis of DNA from rad51, rad52, and rad54 mutants demonstrated that these strains are specifically deficient in the rejoining of radiation-induced DSBs (15, 16, 64). Recent studies have suggested that Rad51, Rad52, Rad54, Rad55, and Rad57 associate to form a multiprotein complex (2, 20, 70). Furthermore, both Rad52 and Rad51, which are conserved in lower and higher eukaryotes, display intrinsic strand-annealing activity (49, 70). Although RAD52 group-mediated homologous recombination is the primary pathway of repair of DSBs in yeast cells, additional pathways have been described. These include nonconservative repair processes which occur in DNA containing direct repeats (e.g., single-strand annealing [30, 56]) and nonrecombinational end-joining pathways, which may be either precise or error prone (9, 13, 39, 44, 45, 47, 68, 72, 78). Genes which have been associated with end-joining pathways include HDF1/YKU70, HDF2/YKU80, RAD50, XRS2, and MRE11. End-joining mechanisms in yeast share several characteristics of DSB repair observed in higher eukaryotes and involve two genes (HDF1/YKU70 and HDF2/YKU80) which are homologs of genes required for DSB repair and V(D)J recombination in mammalian cells (Ku70 and Ku80 [25]). Recent experiments have established that the yeast Ku70 homolog is also essential for maintenance of normal telomere lengths (59). Eukaryotic cells possess checkpoint mechanisms which interrupt cell cycling when chromosomal DNA is damaged. This arrest ensures that DNA repair is completed before downstream cycling events are initiated. Exposure of yeast cells to ionizing radiation or methyl methanesulfonate causes growing cells to arrest in G2. Arrested cells retain large buds and a single undivided nucleus (55, 71, 80, 82). Initial experiments established that rad9 strains were deficient in DNA damage-induced cell cycle arrest (80), and additional genes were subsequently identified (71). Direct evidence that DSBs can initiate the arrest of yeast cells has been obtained from analysis of the consequences of HO endonuclease expression (described below). DSB-induced cell cycle arrest has also been observed in mammalian cells and is dependent upon p53 (50). Recent studies have demonstrated that additional damage-responsive checkpoints in G1 and S phase are present in yeast cells (71). Analysis of DSB repair in yeast has been facilitated by the development of systems for studying the genetic and cytological consequences of a single DSB induced by HO endonuclease. This enzyme stimulates mating-type switching, a gene conversion process, by producing a single DSB at the MAT locus (17, 26, 51). The ends of breaks produced by HO retain complementary 3′ extensions which are 4 nucleotides in length (51). Several groups have used plasmid and chromosomal fusions containing the HO gene under the control of a galactose-regulated promoter to examine effects of DSBs on viability, chromosome metabolism, and cell division. Processing of breaks occurring at MAT has been studied (see, e.g., references 12, 26, and 47), as well as at synthetic HO target sites located on other chromosomes (see, e.g., references 6, 7, 43, 52, 62, and 66). In addition, the consequences of HO expression in radiation-sensitive mutants have been investigated (14, 18, 28, 40, 42, 74, 79). These experiments revealed that HO-induced strand breaks have several of the characteristics of damage produced by ionizing radiation treatment of cells. Such DSBs are recombinogenic, increase aneuploidy and mutagenesis, can arrest cell cycling in G2, and inhibit the growth of radiation-sensitive RAD52 group mutants. High-level expression of two other endonucleases, the restriction enzyme EcoRI and mitochondrial endonuclease I-SceI, has also been examined in yeast cells. Expression of EcoRI in vivo resulted in the formation of DSBs at its target sequence (G∧AATTC) and inhibition of the growth of Rad+ and rad52 cells (3). A single DSB produced by I-SceI efficiently induced homologous recombination in a plasmid containing its recognition sequence (58). While investigating the genetic consequences of enzymatically induced DSBs containing cohesive ends (EcoRI and HO) or blunt ends (PvuII), we confirmed that expression of EcoRI inhibits the growth of rad52 cells. However, most Rad+ strains which were tested grew after the induction of plasmid or chromosomal GAL1::EcoRI fusions. EcoRI-induced DSBs inhibited the growth of rad52 strains and arrested cell division in the G2 phase. Remarkably, rad52 mutants were not more sensitive to killing than wild-type cells. Thus, RAD52 was required for the growth of cells continuously expressing the endonuclease but not for precise repair of induced DSBs after EcoRI synthesis was repressed. In contrast, strains deficient in Ku70-mediated end joining or RAD9-dependent cell cycle checkpoint responses were hypersensitive to killing by EcoRI.