Genome variation profiling by genome tiling array Genome variation profiling by SNP array
Summary
DNA replication stress (DRS)-linked genomic instability has emerged as an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability and phenotypic evolution, we mapped chromosomal alterations in a yeast strain with lowered expression of the replicative DNA polymerase δ. At a whole-genome level, we identified both hotspots of mitotic recombination and chromosome-specific aneuploidy dependent on decreased levels of DNA polymerase δ. The high rate of chromosome loss is likely a reflection of reduced DNA repair capacity in strains with low levels of DNA polymerase. Most recombinogenic DNA lesions were introduced during S or G2 phase, presumably as a consequence of broken replication forks.
Overall design
To reduce the expression of gene POL3, a KANMX-GAL1 cassette was inserted before the ORF of this gene in haploid strains JSC54-1 (W303-1A) and JSC20-1 (YJM789), resulting in mutants DZ1 and DZ2 (genotypes of these strains were listed in Table S1 and primers used to for strain construction were in Table S2), respectively. Mating DZ1 and DZ2 and subsequent knockout of MATalpha generated a diploid strain DZ12. We grew single cells of strain DZ12 to colonies on solid low-gal (2% yeast extract, 2% peptone and 3% raffinose, 0.005% galactose) medium and 35 colonies were selected for whole genome SNP-microarray analysis that can examine about 13,000 SNPs located throughout the genome. 6 DZ12-derived colonies (DZHG1-6) from single cycle growth on high-gal medium (0.05% galactose) were also examined. To induce reciprocal crossover events in DZ12, DZ12 cells were incubated in YPD liquid medium for 6 h to deplete polymerase δ and then plated on solid medium to form sectored colonies (S1-S60). These sectored colonies were analyzed with SNP microarray specific for chromosome 4.