Modeling cancer-associated mutations reveals an integrated role for the Pol epsilon catalytic core in replisome assembly and DNA synthesis
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ABSTRACT:
PROVIDER: PRJNA548119 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9259110_1.fastq.gz | Fastqsanger.gz | |||
| SRR9259110_2.fastq.gz | Fastqsanger.gz | |||
| SRR9259111_1.fastq.gz | Fastqsanger.gz | |||
| SRR9259111_2.fastq.gz | Fastqsanger.gz | |||
| SRR9259106_1.fastq.gz | Fastqsanger.gz |
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Project description:DNA polymerase epsilon (Pol epsilon) plays multiple roles in genome duplication and its catalytic subunit POLE suppresses tumorigenesis in humans. Despite its importance, we have limited understanding of Pol epsilon functional mechanisms and how its loss-of-function affects the evolution of cancer genomes. To address this issue, we used the budding yeast ortholog Pol2 to model cancer-associated POLE mutations that affect an uncharacterized yet highly conserved Pol2/POLE family-specific region. Analysis of pol2 mutants revealed an unexpected role for this region in the incorporation of Pol epsilon into the pre-loading complex during replisome assembly. Cellular and reconstitution experiments further identified a separate role for this region in promoting Pol epsilon polymerase activity. Unlike previously reported POLE mutations leading to hypermutation in cancer cells, the examined pol2 variants increase gross chromosomal rearrangements but not mutation rates. Our findings thus suggest that the Pol epsilon catalytic core integrates replisome assembly and DNA polymerization functions and that POLE tumor suppressive roles likely extend beyond limiting replication errors.
2020-03-29 | GSE132450 | GEO
Project description:The replisome and RNA polymerase II (Pol II) use the sample DNA template during the S phase. Inevitably, there is a conflict between these two machineries when they try to pass each other to accomplish their respective tasks. How such a conflict is resolved, especially in mammalian cells, is a quintessential biological question of which we only have a limited understanding. Using several recently developed techniques, we determined in vivo the replication fork speed, direction, and interruption when the replisome met Pol II. Based on nsRNA-seq of the mouse embryonic fibroblasts (MEFs), we revealed that a tiny fraction of all the genes, i.e., about 1.5 %, downregulated their transcription during the S-phase compared to the G1 phase. A moderate 20% reduction of II density in the S-phase-enriched MEFs compared to asynchronous MEFs was observed. These two means represent the means of temporal segregation from replication. A moderate and significant bias (about 17% difference) was found favoring the co-directional forks over head-on forks. The encounter between the replisome and Pol II was quite eventful. About 21% of the forks were aborted in the Pol II-dense TSS region. Although the majority of the forks could still pass the TSS region, they hesitated and slowed down when the tips of the forks approached the Pol II dense region. Paradoxically, the forks traveled faster in genome regions dotted with more Pol IIs as long as their tips were not near the latter, probably due to a higher degree of chromatin negative supercoiling near Pol IIs. Our study demonstrated that the mammalian cells relied on multiple measures, such as temporal segregation, co-direction preference, and fork abortion, to mitigate the danger of replication-transcription conflict, but all these measures operated only as auxiliaries. The majority of the forks could pass Pol IIs unscathed, indicating the existence of a so-far unidentified, very efficient conflict resolution mechanism.
2026-03-25 | GSE297866 | GEO
Project description:Conflict resolution between the replisome and Pol II
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Project description:The replisome and RNA polymerase II (Pol II) use the sample DNA template during the S phase. Inevitably, there is a conflict between these two machineries when they try to pass each other to accomplish their respective tasks. How such a conflict is resolved, especially in mammalian cells, is a quintessential biological question of which we only have a limited understanding. Using several recently developed techniques, we determined in vivo the replication fork speed, direction, and interruption when the replisome met Pol II. Based on nsRNA-seq of the mouse embryonic fibroblasts (MEFs), we revealed that a tiny fraction of all the genes, i.e., about 1.5 %, downregulated their transcription during the S-phase compared to the G1 phase. A moderate 20% reduction of II density in the S-phase-enriched MEFs compared to asynchronous MEFs was observed. These two means represent the means of temporal segregation from replication. A moderate and significant bias (about 17% difference) was found favoring the co-directional forks over head-on forks. The encounter between the replisome and Pol II was quite eventful. About 21% of the forks were aborted in the Pol II-dense TSS region. Although the majority of the forks could still pass the TSS region, they hesitated and slowed down when the tips of the forks approached the Pol II dense region. Paradoxically, the forks traveled faster in genome regions dotted with more Pol IIs as long as their tips were not near the latter, probably due to a higher degree of chromatin negative supercoiling near Pol IIs. Our study demonstrated that the mammalian cells relied on multiple measures, such as temporal segregation, co-direction preference, and fork abortion, to mitigate the danger of replication-transcription conflict, but all these measures operated only as auxiliaries. The majority of the forks could pass Pol IIs unscathed, indicating the existence of a so-far unidentified, very efficient conflict resolution mechanism.
2026-03-25 | GSE297867 | GEO
Project description:The replisome and RNA polymerase II (Pol II) use the sample DNA template during the S phase. Inevitably, there is a conflict between these two machineries when they try to pass each other to accomplish their respective tasks. How such a conflict is resolved, especially in mammalian cells, is a quintessential biological question of which we only have a limited understanding. Using several recently developed techniques, we determined in vivo the replication fork speed, direction, and interruption when the replisome met Pol II. Based on nsRNA-seq of the mouse embryonic fibroblasts (MEFs), we revealed that a tiny fraction of all the genes, i.e., about 1.5 %, downregulated their transcription during the S-phase compared to the G1 phase. A moderate 20% reduction of II density in the S-phase-enriched MEFs compared to asynchronous MEFs was observed. These two means represent the means of temporal segregation from replication. A moderate and significant bias (about 17% difference) was found favoring the co-directional forks over head-on forks. The encounter between the replisome and Pol II was quite eventful. About 21% of the forks were aborted in the Pol II-dense TSS region. Although the majority of the forks could still pass the TSS region, they hesitated and slowed down when the tips of the forks approached the Pol II dense region. Paradoxically, the forks traveled faster in genome regions dotted with more Pol IIs as long as their tips were not near the latter, probably due to a higher degree of chromatin negative supercoiling near Pol IIs. Our study demonstrated that the mammalian cells relied on multiple measures, such as temporal segregation, co-direction preference, and fork abortion, to mitigate the danger of replication-transcription conflict, but all these measures operated only as auxiliaries. The majority of the forks could pass Pol IIs unscathed, indicating the existence of a so-far unidentified, very efficient conflict resolution mechanism.
2026-03-25 | GSE298050 | GEO
Project description:What is the impact of DNA pol epsilon deficiency on gene expression as well as on the expression of ncRNA, gene silencing? To further investigate the effects of DPB2 over-expression, the transcriptome of DPB2OE lines 1 and 3 was compared to that of wild-type plants by RNA sequencing. Plantlets were grown in vitro on half strength MS for 9 days and used for total RNA extraction. mRNA seq on wild-type Col-0, two DPB2 overexpression lines and one abo4-1 mutant knock out.
2016-07-22 | E-GEOD-71002 | biostudies-arrayexpress
Project description:What is the impact of DNA pol epsilon deficiency on gene expression as well as on the expression of ncRNA, gene silencing etc.? Plantlets were grown in vitro on half strength MS for 9 days and used for total RNA extraction.
2016-07-22 | GSE71002 | 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 | 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:Conflict resolution between the replisome and Pol II [bTMP-seq]
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