Similar Datasets
Project description:Yeast Saccharomyces cerevisiae has been widely used as a model system for studying genome instability. Here, a heterozygous diploid S. cerevisiae strain DZP2 was generated to determine the genomic alterations induced by DNA polymerase ε. The expression of POL2 was regulated by the GAL1 promoter. In combination of a custom SNP microarray, the patterns of chromosomal instability induced by low Pol ε could be explored at a whole genome level in DZP2. Using this system, we found hundreds-fold higher rate of genomic alterations, including aneuploidy, loss of heterozygosity (LOH), and chromosomal rearrangement. 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 rDNA cluster and reduced the length of telomeres. These short telomeres led to 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.
2021-10-07 | GSE185365 | GEO
Project description:Yeast Saccharomyces cerevisiae has been widely used as a model system for studying genome instability. Here, a heterozygous diploid S. cerevisiae strain DZP2 was generated to determine the genomic alterations induced by DNA polymerase ε. The expression of POL2 was regulated by the GAL1 promoter. In combination of a custom SNP microarray, the patterns of chromosomal instability induced by low Pol ε could be explored at a whole genome level in DZP2. Using this system, we found hundreds-fold higher rate of genomic alterations, including aneuploidy, loss of heterozygosity (LOH), and chromosomal rearrangement. 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 rDNA cluster and reduced the length of telomeres. These short telomeres led to 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.
2021-10-07 | GSE185364 | GEO
Project description:Global genomic instability caused by reduced expression of DNA polymerase ε in yeast
| PRJNA768812 | ENA
Project description:Global genomic instability caused by reduced expression of DNA polymerase ε in yeast [DZP2-derived isolates]
| PRJNA768823 | ENA
Project description:Global genomic instability caused by reduced expression of DNA polymerase ε in yeast [DZP2-derived sectored colonies]
| PRJNA768822 | ENA
Project description:Specific regions of genomes (fragile sites) are hot spots for the chromosome rearrangements that are associated with many types of cancer cells. Understanding the molecular mechanisms regulating the stability of chromosome fragile sites, therefore, has important implications in cancer biology. We previously identified two chromosome fragile sites in Saccharomyces cerevisiae that were induced in response to the reduced expression of Pol1p, the catalytic subunit of DNA polymerase alpha. In the study presented here, we show that reduced levels of Pol3p, the catalytic subunit of DNA polymerase delta, induce instability at these same sites and lead to the generation of a variety of chromosomal aberrations. These findings demonstrate that a change in the stoichiometry of replicative DNA polymerases results in recombinogenic DNA lesions, presumably double-strand DNA breaks.
| S-EPMC2519721 | biostudies-literature
Project description:DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G2 phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.
| S-EPMC5167146 | biostudies-literature
Project description:DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.
| S-EPMC3986358 | biostudies-literature
Project description:Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase epsilon bypasses a model abasic site with comparable efficiency to Pol eta and Dpo4, two translesion polymerases. DNA polymerase epsilon also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase epsilon with 3'-5' proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.
| S-EPMC2797225 | biostudies-literature
Project description:DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits--Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1-1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2(ins)) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.
| S-EPMC4061903 | biostudies-literature