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Expression of the cancer-associated DNA polymerase epsilon P286R in fission yeast leads to translesion polymerase dependent hypermutation and defective DNA replication

ABSTRACT: Somatic mutations in the proofreading domain of the replicative DNA polymerase epsilon (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, microsatellite stability and a unique mutational signature, with predominance of C > A transversions in the context TCT. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R also has a strong mutator effect in vivo, with high frequency of base substitutions and relatively few frameshift mutations. The mutations are equally distributed across different genomic regions, but they occur within an AT-rich context. The most abundant mutations are TCT > TAT transversions and, in contrast to human, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The high mutation burden of pol2-P287R leads to increased sensitivity to elevated dNTP levels and DNA damaging agents and a phenotype that is exacerbated by depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of persistent ssDNA or DNA damage, and pol2-P287R shows synthetic lethality with rad3 deletion, indicative of checkpoint activation. Significantly, deletion of translesion polymerases kappa and eta partially suppresses pol2-P287R hypermutation, indicating that both enzymes contribute to this phenotype.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE169231 | GEO | 2021/06/11

REPOSITORIES: GEO

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Modeling cancer-associated mutations reveals an integrated role for the Pol epsilon catalytic core in replisome assembly and DNA synthesis

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Immunoglobulin enhancers increase RNA polymerase 2 stalling at somatic hypermutation target sequences [GRO-seq]

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RNA Polymerase II interactome in presence or absence of SPT6 in U2OS cells

Project description:SPT6 is a histone chaperone that tightly binds RNA polymerase II (POL2) during transcription elongation. However, its primary role in POL2 transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in POL2 behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for POL2 processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in POL2 termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the absence of SPT6. In conclusion, targeted protein degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and POL2 elongation factor.

2021-06-28 | PXD025243 | Pride

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Global genomic instability caused by reduced expression of DNA polymerase ε in yeast [DZP2-derived isolates]

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.

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