RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase δ

Fortune, John M.; Stith, Carrie M.; Kissling, Grace E.; Burgers, Peter M.; Kunkel, Thomas A.

doi:10.1093/nar/gkl403

Cited by 68 publications

(70 citation statements)

References 40 publications

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“…The S-phase dNTP concentrations were calculated using measurements of dNTP levels in wild-type and pol3-R696W haploids containing pPOL3, as described in Table 3. Similar to other studies (8,44), the frequency of mutations resulting from synthesis by wild-type Polδ was not significantly different from the background mutation frequency for unfilled M13mp2 gapped substrate, regardless of whether the S-phase or standard 100 μM dNTP concentration was used (Table 2). In contrast, DNA synthesis by Polδ-R696W was highly error-prone.…”

Section: Resultssupporting

confidence: 85%

“…Yeast 2μ-based plasmids expressing POL3 from the GAL1 promoter, pBL336 (83), pBL335, and pBL335-5DV (44), which are referred to as pPOL3, pPOL3.GST, and pPOL3.GST-5DV in this study, were provided by Peter Burgers (Washington University School of Medicine, St. Louis, MO). Plasmids pPOL3.GST-R696W and pPOL3.GST-5DV,R696W were constructed by gap repair in yeast strains, in which the effects of these plasmids were studied.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

Mertz

Sharma²,

Chabes

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Defects in DNA polymerases δ (Polδ) and e (Pole) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.DNA polymerase δ | colon cancer | dNTP pools | mutagenesis | DNA replication fidelity W hen functioning properly, DNA replication is phenomenally accurate, making ∼10 −10 mutations per base pair during each replication cycle (1). In respect to human biology, it means that, on average, less than one mutation occurs each time the human genome is replicated. This amazing exactitude is contingent upon the serial action of DNA polymerase selectivity, exonucleolytic proofreading, and DNA mismatch repair (MMR). MMR defects increase spontaneous mutagenesis in numerous model systems (2) and give rise to cancer in mice (3). In humans, inherited mutations in MMR genes predispose to colorectal cancer (CRC) in Lynch syndrome (4, 5). Additionally, MMR genes are inactivated via hypermethylation in ∼15% of sporadic CRC, endometrial cancer (EC), and gastric cancer (6).Like defects in MMR, mutations that decrease the base selectivity or proofreading activity of replicative DNA polymerases elevate spontaneous mutagenesis in eukaryotic cells (7-14) and cancer incidence in mice (15)(16)(17)(18). Germline mutations affecting the exonuclease domains of DNA polymerases δ (Polδ) and e (Pole) cause hereditary CRC (19), and somatic changes in the exonuclease domain of Pole were found in sporad...

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Section: Resultssupporting

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

Mertz

Sharma²,

Chabes

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…34) was used for overproduction and purification of GSTtagged Rev1 fragments. Strains used in the UV sensitivity assays are described in Table 1.…”

Section: Methodsmentioning

confidence: 99%

The Non-canonical Protein Binding Site at the Monomer-Monomer Interface of Yeast Proliferating Cell Nuclear Antigen (PCNA) Regulates the Rev1-PCNA Interaction and Polζ/Rev1-dependent Translesion DNA Synthesis

Sharma¹,

Kochenova

Shcherbakova

2011

Journal of Biological Chemistry

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Rev1 and DNA polymerase (Pol) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Pol lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Pol with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pulldown and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Pol-dependent TLS pathway. Taken together, the data suggest that Pol and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.Cellular DNA is continuously attacked by endogenous and exogenous agents that damage the bases and the DNA backbone. Damage that is not repaired prior to the S phase of cell cycle can block the replication machinery, because most lesions cannot be accommodated in the highly selective active site of replicative DNA polymerases (1, 2). Replication stalling activates several damage tolerance mechanisms that help bypass the damage in either an accurate or mutagenic manner. Translesion DNA synthesis (TLS) 3 is an important damage tolerance pathway, in which specialized DNA polymerases are recruited to synthesize DNA through template lesions (3). Structural studies showed that TLS polymerases have a more open active site that allows them to accommodate a variety of DNA lesions and catalyze polymerization on damaged templates (4). The accuracy of TLS can vary depending on the particular lesion and the DNA polymerases involved. It is, however, an inherently mutagenic process, because the damage can alter the coding properties of the bases, and because TLS polymerases generally have low fidelity (2).In human cells, TLS polymerases include Y family enzymes Pol, Pol, Pol, and REV1, and the B family enzyme Pol. Although the Y family polymerases share little amino acid sequence similarity with the classical DNA polymerases, they have a similar overall "right-hand" architecture with the "palm," "thumb," and "fingers" domains. The thumb and fingers domains of the Y family enzymes, however, are short and do not make as many contacts wi...

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“…These observations demonstrate that UL42 plays an important role in DNA replication fidelity. Although PCNA has been linked to replication fidelity in yeast, PCNA and gp45 have been linked to DNA replication fidelity in vitro (2,3,5,7,8,24), and UL42 can increase fidelity in vitro (4), to our knowledge, this is the first study to demonstrate an effect of a processivity factor in the replication fidelity of a virus in mammalian cells.…”

Section: Discussionmentioning

confidence: 98%

“…Mutations affecting PCNA can lead to elevated mutation rates in Saccharomyces cerevisiae (5,7). In vitro studies have demonstrated that the addition of PCNA can increase misincorporation errors by mammalian Pol ␦ on defined primer/templates (24) and reduce the rate of deletion mutations during the synthesis of repeated sequences by yeast Pol ␦ (8). T4 gp45 also can influence replication fidelity in vitro (2,3).…”

mentioning

confidence: 99%

Mutations That Decrease DNA Binding of the Processivity Factor of the Herpes Simplex Virus DNA Polymerase Reduce Viral Yield, Alter the Kinetics of Viral DNA Replication, and Decrease the Fidelity of DNA Replication

Jiang

Hwang

Randell

et al. 2007

J Virol

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The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol-and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.

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RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase δ

Cited by 68 publications

References 40 publications

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

The Non-canonical Protein Binding Site at the Monomer-Monomer Interface of Yeast Proliferating Cell Nuclear Antigen (PCNA) Regulates the Rev1-PCNA Interaction and Polζ/Rev1-dependent Translesion DNA Synthesis

Mutations That Decrease DNA Binding of the Processivity Factor of the Herpes Simplex Virus DNA Polymerase Reduce Viral Yield, Alter the Kinetics of Viral DNA Replication, and Decrease the Fidelity of DNA Replication

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