Ke Zhang, Yang Sui, Wu-Long Li, Gen Chen, Xue-Chang Wu, Robert J. Kokoska, Thomas D. Petes and Dao-Qiong Zheng.

PNAS March 15, 2022 | 119 (12) e2119588119

Significance

Although most studies of the genetic regulation of genome stability involve an analysis of mutations within the coding sequences of genes required for DNA replication or DNA repair, recent studies in yeast show that reduced levels of wild-type enzymes can also produce a mutator phenotype. By whole-genome sequencing and other methods, we find that reduced levels of the wild-type DNA polymerase ε in yeast greatly increase the rates of mitotic recombination, aneuploidy, and single-base mutations. The observed pattern of genome instability is different from those observed in yeast strains with reduced levels of the other replicative DNA polymerases, Pol α and Pol δ. These observations are relevant to our understanding of cancer and other diseases associated with genetic instability.

Abstract

DNA polymerase ε (Pol ε) is one of the three replicative eukaryotic DNA polymerases. Pol ε deficiency leads to genomic instability and multiple human diseases. Here, we explored global genomic alterations in yeast strains with reduced expression of POL2, the gene that encodes the catalytic subunit of Pol ε. Using whole-genome SNP microarray and sequencing, we found that low levels of Pol ε elevated the rates of mitotic recombination and chromosomal aneuploidy by two orders of magnitude. Strikingly, low levels of Pol ε resulted in a contraction of the number of repeats in the ribosomal DNA cluster and reduced the length of telomeres. These strains also had an elevated frequency of break-induced replication, resulting in terminal loss of heterozygosity. In addition, low levels of Pol ε increased the rate of single-base mutations by 13-fold by a Pol ζ-dependent pathway. Finally, the patterns of genomic alterations caused by low levels of Pol ε were different from those observed in strains with low levels of the other replicative DNA polymerases, Pol α and Pol δ, providing further insights into the different roles of the B-family DNA polymerases in maintaining genomic stability.

See https://www.pnas.org/doi/10.1073/pnas.2119588119

Figure 1: Levels of Pol ε and growth rates of GAL1-POL2 strains in media containing different concentrations of galactose. (A) The relative mRNA levels of POL2 in strains JSC25 (wild-type) and DZP2 (GAL1-POL2). RNA was isolated from these strains grown in HG, LG, and no-galactose medium (YPD) (details in Materials and Methods). Levels of POL2-specific mRNA were normalized to the levels of ACT1-specific mRNA. (B) Western blot analysis of the levels of the Pol2p in JSC25 and DZP2 incubated in various types of media. The level of GAPDH was used as a control. (C) Comparative growth rates of JSC25 and DPZ2 on HG, LG, and YPD plates for strains JSC25 and DZP2. Ten-fold dilutions of each strain were spotted onto solid medium and grown at 30 °C.