DNA polymerase ε relies on a unique domain for efficient replisome assembly and strand synthesis

Meng, Xiang−Zhou; Wei, Lei; Devbhandari, Sujan; Zhang, Tuo; Xiang, Jenny; Remus, Dirk; Zhao, Xiaolan

doi:10.1038/s41467-020-16095-x

Cited by 18 publications

(30 citation statements)

References 57 publications

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“…2D gel analysis was performed as previously described (Meng et al 2020). Briefly, log-phase yeast cultures were treated with 5 µg/mL α factor (Bio Basic) until at least 90% of cells exhibited G1 arrest.…”

Section: D Agarose Gel Electrophoresismentioning

confidence: 99%

Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance

Bonner

Wan

et al. 2021

Genes Dev.

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SUMO modification regulates diverse cellular processes by targeting hundreds of proteins. However, the limited number of sumoylation enzymes raises the question of how such a large number of substrates are efficiently modified. Specifically, how genome maintenance factors are dynamically sumoylated at DNA replication and repair sites to modulate their functions is poorly understood. Here, we demonstrate a role for the conserved yeast Esc2 protein in this process by acting as a SUMO E2 cofactor. Esc2 is required for genome stability and binds to Holliday junctions and replication fork structures. Our targeted screen found that Esc2 promotes the sumoylation of a Holliday junction dissolution complex and specific replisome proteins. Esc2 does not elicit these effects via stable interactions with substrates or their common SUMO E3. Rather, we show that a SUMO-like domain of Esc2 stimulates sumoylation by exploiting a noncovalent SUMO binding site on the E2 enzyme. This role of Esc2 in sumoylation is required for Holliday junction clearance and genome stability. Our findings thus suggest that Esc2 acts as a SUMO E2 cofactor at distinct DNA structures to promote the sumoylation of specific substrates and genome maintenance.

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Section: D Agarose Gel Electrophoresismentioning

confidence: 99%

Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance

Bonner

Wan

et al. 2021

Genes Dev.

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“…Table 1; 40% decrease of mutagenesis) is less pronounced than for other mutations leading to a defective replisome, including the pol2-1 allele where the POL2 ORF is disrupted in the middle by the URA3 marker (>80% reduction of mutagenesis) [41]. The reasons for these peculiarities are still unclear and might reflect the multifaceted role of Pol2 and its catalytically active domain in the replication fork [75]. In addition to the elevation of mutation rate, pol2rc-ΔN strains exhibit a high rate of illegitimate mating, which results from chromosomal rearrangements and losses, as well as some not clearly defined "primary" DNA lesions temporarily affecting MAT locus transcription [47,49].…”

Section: Discussionmentioning

confidence: 98%

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations inCDC28gene

Stepchenkova

Zhuk

Cui

et al. 2020

Preprint

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DNA polymerase ε (pol ε) participates in the leading DNA strand synthesis in eukaryotes. The catalytic subunit of this enzyme, Pol2, is a fusion of two ancestral B-family DNA polymerases. Paradoxically, the catalytically active N-terminal pol is dispensable, and an inactive C-terminal pol is essential for yeast cell viability. Despite extensive studies of strains without the active N-terminal part (mutation pol2-16), it is still unclear how they survive and what the mechanism is of rapid recovery of initially miserably growing cells. Slow progress is attributed to the difficultly of obtaining strains with the defect. We designed a robust method for the construction of mutants with only the C-terminal part of Pol2 using allele pol2rc-ΔN optimized for high protein production. Colonies bearing pol2rc-ΔN appear three times sooner than colonies of pol2-16 but exhibit similar growth defects: sensitivity to hydroxyurea, chromosomal instability, and an elevated level of spontaneous mutagenesis. UV-induced mutagenesis is partially affected, it is lower only at high doses in some reporters. The analysis of the genomes of pol2rc-ΔN isolates revealed the prevalence of nonsynonymous mutations suggesting that the growth recovery was a result of evolution by positive selection for better growth fueled by variants produced by the elevated mutation rate. Mutations in the CDC28 gene, the primary regulator of the cell cycle, were repeatedly found in independent clones. Genetic analysis established that cdc28 alleles single-handedly improve the growth of pol2rc-ΔN strains and suppress HU-sensitivity. The affected amino acids are located on the Cdc28 molecule’s surfaces that mediate contacts with cyclins and kinase subunits. Our work establishes the significance of the CDC28 gene for the resilience of replication and predicts that changes in mammalian homologs, cyclin-dependent kinases may play a role in remastering replication to compensate for the defects in the leading strand synthesis by the dedicated polymerase.Author SummaryThe catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable while the inactive C-terminal part is required for viability. The corresponding strains show a severe growth defect, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly produced fast-growing clones. We discovered that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs during evolution by positive selection for a better growth fueled by variants produced by elevated mutation rates. Mutations in the cell cycle-dependent kinase gene, CDC28, can single-handedly improve the growth of strains lacking the N-terminal part of Pol2. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may play a role in response to the defects of active leading strand polymerase.

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“…Polymerase epsilon (POLE) is a replicative polymerase important for efficient replisome assembly and strand synthesis, supporting tumor suppression [81]. POLE is involved in DNA repair and especially methylated cytosines (5mCs) are frequently mutated.…”

Section: Methylation Of the Tp53 Genementioning

confidence: 99%

Epigenetic Alterations Upstream and Downstream of p53 Signaling in Colorectal Carcinoma

Tomičić

Dawood

Efferth

2021

Cancers

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Colorectal cancer (CRC) belongs to the most common tumor types, and half of all CRC harbor missense mutations in the TP53 tumor suppressor gene. In addition to genetically caused loss of function of p53, epigenetic alterations (DNA methylation, histone modifications, micro-RNAs) contribute to CRC development. In this review, we focused on epigenetic alterations related to the entire p53 signaling pathway upstream and downstream of p53. Methylation of genes which activate p53 function has been reported, and methylation of APC and MGMT was associated with increased mutation rates of TP53. The micro-RNA 34a activates TP53 and was methylated in CRC. Proteins that regulate TP53 DNA methylation, mutations, and acetylation of TP53-related histones were methylated in CRC. P53 regulates the activity of numerous downstream proteins. Even if TP53 is not mutated, the function of wildtype p53 may be compromised if corresponding downstream genes are epigenetically inactivated. Thus, the role of p53 for CRC development, therapy response, and survival prognosis of patients may be much more eminent than previously estimated. Therefore, we propose that novel diagnostic devices measuring the entirety of genetic and epigenetic changes in the “p53 signalome” have the potential to improve the predictive and prognostic power in CRC diagnostics and management.

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DNA polymerase ε relies on a unique domain for efficient replisome assembly and strand synthesis

Cited by 18 publications

References 57 publications

Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance

Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations inCDC28gene

Epigenetic Alterations Upstream and Downstream of p53 Signaling in Colorectal Carcinoma

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