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| 2. |
- Chilkova, Olga, et al.
(författare)
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The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA.
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 35:19, s. 6588-6597
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol delta and Pol epsilon in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol epsilon has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol delta has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol delta and Pol epsilon was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol delta and Pol epsilon on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol epsilon when compared to Pol delta. We conclude that Pol epsilon and Pol delta exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.
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| 3. |
- Hogg, Matthew, et al.
(författare)
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Structural basis for processive DNA synthesis by yeast DNA polymerase ε
- 2014
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Ingår i: Nature Structural & Molecular Biology. - : Nature Publishing Group. - 1545-9993 .- 1545-9985. ; 21:1, s. 49-56
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Tidskriftsartikel (refereegranskat)abstract
- DNA polymerase ε (Pol ε) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure of the catalytic core of Pol ε (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ε has the highest fidelity among B-family polymerases despite the absence of an extended b-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ε to encircle the nascent doublestranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes
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| 4. |
- Nick McElhinny, Stephanie A, et al.
(författare)
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Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.
- 2010
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:11, s. 4949-4954
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Tidskriftsartikel (refereegranskat)abstract
- Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36- to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase epsilon, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol delta and Pol alpha, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols alpha, delta, and epsilon, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase epsilon has difficulty bypassing a single rNMP present within a DNA template.
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| 5. |
- Nick McElhinny, Stephanie A, et al.
(författare)
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Genome instability due to ribonucleotide incorporation into DNA
- 2010
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Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 6:10, s. 774-81
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Tidskriftsartikel (refereegranskat)abstract
- Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine whether ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active-site methionine in yeast DNA polymerase ϵ (Pol ϵ). Ribonucleotide incorporation in vitro was three-fold lower for M644L and 11-fold higher for M644G Pol ϵ compared to wild-type Pol ϵ. This hierarchy was recapitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S phase, had elevated dNTP pools and generated 2-5-base-pair deletions in repetitive sequences at a high rate and in a gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair and that defective repair results in replicative stress and genome instability via DNA strand misalignment.
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| 6. |
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| 7. |
- Pursell, Zachary F, et al.
(författare)
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Yeast DNA polymerase epsilon participates in leading-strand DNA replication.
- 2007
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 317:5834, s. 127-130
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Tidskriftsartikel (refereegranskat)abstract
- Multiple DNA polymerases participate in replicating the leading and lagging strands of the eukaryotic nuclear genome. Although 50 years have passed since the first DNA polymerase was discovered, the identity of the major polymerase used for leading-strand replication is uncertain. We constructed a derivative of yeast DNA polymerase ϵ that retains high replication activity but has strongly reduced replication fidelity, particularly for thymine-deoxythymidine 5'-monophosphate (T-dTMP) but not adenine-deoxyadenosine 5'-monophosphate (A-dAMP) mismatches. Yeast strains with this DNA polymerase ϵ allele have elevated rates of T to A substitution mutations. The position and rate of these substitutions depend on the orientation of the mutational reporter and its location relative to origins of DNA replication and reveal a pattern indicating that DNA polymerase ϵ participates in leading-strand DNA replication.
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