Cloning and characterisation of the Schizosaccharomyces pombe rad 32 gene : A gene required for repair of double strand break and recombination

A new Schizosaccharomyces pombe mutant (rad32) which Is sensitive to gamma and UV irradiation is described. Pulsed field gel electrophoresls of DNA from Irradiated cells indicates that the rad32 mutant, in comparison to wild type cells, has decreased ability to repair DNA double strand breaks. The mutant also undergoes decreased melotlc recombination and displays reduced stability of mlnlchromosomes. The rad32 gene has been cloned by complementation of the UV sensitive phenotype. The gene, which is not essential for cell viability and is expressed at a moderate level in mitotically dividing cells, has significant homology to the meiotic recombination gene MRE11 of Saccharomyces cerevisiae. Epistasis analysis indicates that rad32 functions in a pathway which includes the rhp51 gene (the S.pombe homologue to S.cerevlslae RAD51) and that cells deleted for the rad32 gene in conjunction with either the radd deletion (a G2 checkpoint mutation) or the rad2 deletion (a chromosome stability and potential nucleotide excision repair mutation) are not viable. INTRODUCTION DNA repair processes are fundamentally important if cells are to maintain their genetic integrity. Organisms have thus evolved a number of processes by which DNA damage is recognised and repaired, and these include a well characterised nucleotide excision repair pathway and a damage tolerance mechanism involving recombination. Recent studies have begun to yield important data on the mechanisms associated with nucleotide excision repair and the highly conserved nature of the proteins involved (e.g. I -3). In contrast, somewhat less is known about the molecular mechanisms involved in recombination in eukaryotes. The fission yeast, S.pombe, has proved to be a good model system for the study of eukaryotic processes, such as the cell cycle (e.g. 4) and more recently nucleotide excision repair (e.g. 5,6). Radiation sensitive mutants in S.pombe were originally assigned to 23 complementation groups (7,8), although this number has been expanded to include several new G2 checkpoint mutants (9). Categorization of the corresponding genes into epistasis groups has been limited and effective only for those genes involved in nucleotide excision repair and a G2 checkpoint pathway. Of the original S.pombe mutants which are sensitive to gamma irradiation, only radii and rad22 are now categorized as being defective in recombination (10,11). A more recent search for meiotic recombination mutants has led to the identification of 10 new complementation groups, mutants in three of which are sensitive to DNA damaging agents (12). Recombination is not only required for the tolerance of DNA damage, but is also central to meiosis. In S.cerevisiae, recombination mutants have been isolated in a number of screens e.g. the rad52 series of mutants which are sensitive to gamma radiation (e.g. 13) as well as several mutants defective in meiotic recombination (e.g. 14). Recent studies have shown that some of the RAD52 series of genes are highly conserved between the yeasts, S.cerevisiae and Spombe, and higher eukaryotes. e.g. RAD51 (15), RAD52 (16) and RAD54 (17). We report here the isolation and characterization of a new gamma radiation sensitive S.pombe mutant rad32, and the cloning and sequence analysis of the corresponding gene. The mutant is defective in meiotic recombination, displays spontaneous chromosome instability and is defective in repairing DNA double strand breaks. The rad32 gene has homology to the S.cerevisiae meiotic recombination gene MRE11 and is not essential for cell viability. Epistasis analysis indicates that Rad32 is likely to function in a pathway that includes Rhp51 (the S.pombe homologue to S.cerevisiae Rad51). MATERIALS AND METHODS Plasmids, strains and growth conditions The S.pombe plasmid pUR 19 and the genomic library used in this study have been described elsewhere (18). The S.pombe cDNA library was a gift from L. Guarente (19). S.pombe strains used in * To whom correspondence should be addressed at U niersity of Sssex on Jne 8, 2014 http://narrdjournals.org/ D ow nladed from 384 Nucleic Acids Research, 1995, Vol. 23, No 3 this study are shown in Table 1, strain GP24 was a gift from G. Smith (Seattle, 12). Plasmids were grown in E coli strain DH5a, [endAl, hsdR17, (rk~, mkr), supE44, thi-1, recAl, ladqZ-M15 (lacproAB)]. M13 derivatives were grown in DH5of (as DH5a except that it contains an integrated F). Procedures and media used for the routine growth and maintenance of S.pombe strains were as reported in our previous work (20).