Identification of cis and trans factors that regulate genetic stability in Saccharomyces cerevisiae

The genome of an organism is not uniformly mutagenic. The overall aim of this project was to identify cis and trans factors that may contribute to such differential mutagenic activities within the genome using Saccharomyces cerevisiae. A well-characterised recombination reporter construct, hisG-URA3-hisG, was separately introduced into five different locations of chromosome III. Each locus had differing features with respect to their replication dynamics: three replication termination sites, two of which coincided with “Replication Slow Zones” (RSZ; Cha and Kleckner, 2002), one replication origin and a region of no discernable feature. Fluctuation analysis was used to assess the rate of URA3 inactivation at each locus. First, the effects of temperature, a replication inhibitor hydroxyurea, and ploidy were assessed. Significant differences in mutation rates existed in diploid strains heterozygous for the construct in these conditions, but not in respective haploids. The effects of inactivating various genes known to be involved in genome stability were also examined. Elimination of an essential signal transduction protein, Mec1p, or a DNA helicase required for efficient replication, Rrm3p, led to an increase in mutation rates only in diploid strains. No statistically significant effect was seen when a top2 temperature-sensitive allele was used in either haploids or diploids. In general, no cis effect was observed in any of these mutant backgrounds. The nature of genetic alterations associated with URA3 inactivation was also determined by Southern analysis for the five loci. The analysis revealed that the nature of genetic alteration is regulated in a cis manner, as URA3 inactivation was either exclusively via recombination or by small changes depending on the location of the reporter construct. These findings reveal some unexpected ways in which cis and trans factors may regulate mutagenic events in budding yeast. These will be discussed in context of eukaryotic genome instability in general.