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

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

PROVIDER: PRJNA548119 | ENA |

REPOSITORIES: ENA

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

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

Transcriptomic changes associated with DNA pol epsilon deficiency

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

Transcriptomic changes associated with DNA pol epsilon deficiency

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

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.

2021-10-07 | GSE185365 | GEO

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

2021-10-07 | GSE185364 | GEO

Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome.

Project description:Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.

| S-EPMC6265327 | biostudies-literature

CMG-Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome.

Project description:The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.

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Fork Pausing Complex Engages Topoisomerases at the Replisome

Project description:Replication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) acts as a ‘molecular brake’ and promotes fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C-terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a ‘sTOP’ mechanism (‘slowing down with TOPoisomerases I-II’), which we show also contributes to protecting cells from topoisomerase-blocking agents.

2019-11-07 | GSE140034 | GEO

Transcriptomic profiling of Arabidopsis DNA POLYMERASE EPSILON CATALYTIC SUBUNIT A (AtPOL2a) and B (AtPOL2b) mutants

Project description:Transcriptomic profiling of Arabidopsis DNA POLYMERASE EPSILON CATALYTIC SUBUNIT A (AtPOL2a) and B (AtPOL2b) mutants

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Single-molecule mapping of replisome progression

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