EFFECTS OF DNA DOUBLE-STRAND AND SINGLE-STRAND BREAKS ON INTRACHROMOSOMAL RECOMBINATION EVENTS IN CELL-CYCLE-ARRESTED YEAST-CELLS

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of in itiating these events. Two genetic sl stems were constructed in Saccha romyces cerevisiae that select for DEL recombination events between du plicated alleles of CDC28 and TUB2. We determined effects of double-st rand breaks (DSBs) and single-strand breaks (SSBs) between the duplica ted alleles on DEL recombination when induced in dividing cells or cel ls arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gI Ip), respectively. I-Sce I-induced DSBs caused an increase in DEL reco mbination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DS B repair. In contrast. gIIp-induced SSBs caused an increase in DEL rec ombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most re levant lesion, and UV, inducing other forms of DNA damage. UV irradiat ion did not increase DEL recombination frequencies in G1 or G2, wherea s gamma-rays increased DEL recombination frequencies ill both phases. Both forms of radiation, however, induced DEL recombination in dividin g cells. The results suggest that DSBs but not SSBs induce DEL recombi nation, probably via the single-strand annealing pathway. Further, DSB s in dividing cells may result from the replication of a UV or SSB-dam aged template. Alternatively, UV induced events may occur by replicati on slippage after DNA polymerase pausing in front of the damage.