Human DNA Polymerase ϵ Is Able to Efficiently Extend from Multiple Consecutive Ribonucleotides

Göksenin, A. Yasemin; Zahurancik, Walter J.; LeCompte, Kimberly G.; Taggart, David; Suo, Zucai; Pursell, Zachary F.

doi:10.1074/jbc.m112.422733

Cited by 52 publications

(63 citation statements)

References 49 publications

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“…The small differences in exonuclease rates are in agreement with in vitro fidelity measurements on gapped substrates that showed that the two forms of Pol ⑀ have comparable levels of fidelity (45). The exonucleolytic rate obtained for yeast Pol2 core and Pol ⑀ (ϳ11 s Ϫ1 at 25°C) on a correctly base-paired primer-template are more than 10-fold higher than the reported value for the human Pol2 core (0.86 s Ϫ1 at 37°C) (46). The difference between the studies might in part be due to differences in the design of the experiments as well as to differences in sequence context.…”

Section: Discussionsupporting

confidence: 64%

Yeast DNA Polymerase ϵ Catalytic Core and Holoenzyme Have Comparable Catalytic Rates

Ganai

Osterman

Johansson

2015

Journal of Biological Chemistry

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Background:The catalytic rates for yeast Pol ⑀ are unknown. Results: The catalytic core and holoenzyme have comparable catalytic rates, but the loading onto primer termini differs. Conclusion:The accessory subunits and C terminus of the catalytic subunit do not influence the catalytic rates. Significance: The catalytic rates of Pol ⑀ provide a benchmark for future mechanistic studies of leading strand synthesis.

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Section: Discussionsupporting

confidence: 64%

Yeast DNA Polymerase ϵ Catalytic Core and Holoenzyme Have Comparable Catalytic Rates

Ganai

Osterman

Johansson

2015

Journal of Biological Chemistry

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“…The increased number of errors in the dpb2-100 mutant could be caused by an increased incorporation of ribonucleotides, since Pol has previously been shown to incorporate ribonucleotides efficiently [65][66][67][68]. To check this possibility, we investigated whether the deletion of rnh201 (a component of RER-ribonucleotide excision repair) disturbs the removal of ribonucleotides from DNA, increasing the rate of mutations in the dpb2-100 strain.…”

Section: Errors In Dpb2-100 Strain Are Not Due To Increased Incorporamentioning

confidence: 98%

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex

et al. 2015

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DNA polymerase epsilon interacts with the CMG (Cdc45-MCM-GINS) complex by Dpb2p, the non-catalytic subunit of DNA polymerase epsilon. It is postulated that CMG is responsible for targeting of Pol ɛ to the leading strand. We isolated a mutator dpb2-100 allele which encodes the mutant form of Dpb2p. We showed previously that Dpb2-100p has impaired interactions with Pol2p, the catalytic subunit of Pol ɛ. Here, we present that Dpb2-100p has strongly impaired interaction with the Psf1 and Psf3 subunits of the GINS complex. Our in vitro results suggest that while dpb2-100 does not alter Pol ɛ's biochemical properties including catalytic efficiency, processivity or proofreading activity - it moderately decreases the fidelity of DNA synthesis. As the in vitro results did not explain the strong in vivo mutator effect of the dpb2-100 allele we analyzed the mutation spectrum in vivo. The analysis of the mutation rates in the dpb2-100 mutant indicated an increased participation of the error-prone DNA polymerase zeta in replication. However, even in the absence of Pol ζ activity the presence of the dpb2-100 allele was mutagenic, indicating that a significant part of mutagenesis is Pol ζ-independent. A strong synergistic mutator effect observed for transversions in the triple mutant dpb2-100 pol2-4 rev3Δ as compared to pol2-4 rev3Δ and dpb2-100 rev3Δ suggests that in the presence of the dpb2-100 allele the number of replication errors is enhanced. We hypothesize that in the dpb2-100 strain, where the interaction between Pol ɛ and GINS is weakened, the access of Pol δ to the leading strand may be increased. The increased participation of Pol δ on the leading strand in the dpb2-100 mutant may explain the synergistic mutator effect observed in the dpb2-100 pol3-5DV double mutant.

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“…We used site-directed mutagenesis to introduce several CRC and endometrial cancer mutations into our previously described POLE expression construct (Korona et al 2011;Goksenin et al 2012), containing all six conserved motifs necessary for 39-59 exonuclease and DNA synthesis activities (Supplemental Fig. S3A).…”

Section: Mutation Of Pole-exo Impairs 39-59 Exonuclease Activitymentioning

confidence: 99%

“…Expression and purification of recombinant cancer-associated human POLE constructs An expression vector encoding amino acid residues 1-1189 of the exonuclease-proficient catalytic subunit of human POLE (POLE-N140) was used as described (Korona et al 2011;Goksenin et al 2012). Site-directed mutagenesis was used to introduce the POLEexo* mutations encoding: P286H, F367S, and S459F substitutions.…”

Section: Mutation Context Clustering and Enrichmentmentioning

confidence: 99%

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication

et al. 2014

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Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT!TAT and TCG!TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C!A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

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Human DNA Polymerase ϵ Is Able to Efficiently Extend from Multiple Consecutive Ribonucleotides

Cited by 52 publications

References 49 publications

Yeast DNA Polymerase ϵ Catalytic Core and Holoenzyme Have Comparable Catalytic Rates

Yeast DNA Polymerase ϵ Catalytic Core and Holoenzyme Have Comparable Catalytic Rates

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication

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