Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Both in yeast and humans, DNA polymerase (Pol) (eta) functions in the error-free replication of UV-damaged DNA, and Pol(eta) has the unique ability to efficiently replicate through a cis-syn thymine-thymine (T-T) dimer by inserting two As opposite the two Ts of the dimer. Although human DINB1-encoded Pol(kappa) belongs to the same protein family as Pol(eta), Pol(kappa) shows no ability to bypass this DNA lesion and its biological function has remained unclear. Here, we examine Pol(kappa) for its ability to extend from primer-terminal mispairs opposite nondamaged and damaged DNA templates. We find that Pol(kappa) is a promiscuous extender of primer-terminal mispairs opposite nondamaged DNA templates, and interestingly, it is also very efficient at extending from a G opposite the 3'T of a T-T dimer. These observations provide biochemical evidence for a role of Pol(kappa) in the extension of mismatched base pairs during normal DNA replication, and in addition, they implicate Pol(kappa) in the mutagenic bypass of T-T dimers. In its proficient mismatch extension ability, Pol(kappa) is more similar to the unrelated DNA polymerase zeta than it is to the phylogenetically related Pol(eta) or Pol(iota). Thus, in humans, Pol(kappa) would compete with Pol(zeta) for the extension of mismatched base pairs on damaged and undamaged DNAs.
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PMID:Human DINB1-encoded DNA polymerase kappa is a promiscuous extender of mispaired primer termini. 1184 89

Rev1 protein of Saccharomyces cerevisiae functions with DNA polymerase zeta in mutagenic trans-lesion synthesis. Because of the reported preferential incorporation of a C residue opposite an abasic site, Rev1 has been referred to as a deoxycytidyltransferase. Here, we use steady-state kinetics to examine nucleotide incorporation by Rev1 opposite undamaged and damaged template residues. We show that Rev1 specifically inserts a C residue opposite template G, and it is approximately 25-, 40-, and 400-fold less efficient at inserting a C residue opposite an abasic site, an O(6)-methylguanine, and an 8-oxoguanine lesion, respectively. Rev1 misincorporates G, A, and T residues opposite template G with a frequency of approximately 10(-3) to 10(-4). Consistent with this finding, Rev1 replicates DNA containing a string of Gs in a template-specific manner, but it has a low processivity incorporating 1.6 nucleotides per DNA binding event on the average. From these observations, we infer that Rev1 is a G template-specific DNA polymerase.
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PMID:Yeast Rev1 protein is a G template-specific DNA polymerase. 1185 Apr 24

REV1 functions in the DNA polymerase zeta mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3'-->5' proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19-27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N2-dG, (-)-trans-anti-benzo[a]pyrene-N2-dG and 1,N6-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6-4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase kappa, bypass of the trans-anti-benzo[a]pyrene-N2-dG adducts and the 1,N6-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass.
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PMID:Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion. 1191 24

Information about the mechanisms that generate mutations in eukaryotes is likely to be useful for understanding human health concerns, such as genotoxicity and cancer. Eukaryotic mutagenesis is largely the outcome of attacks by endogenous and environmental agents. Except for DNA repair, cell cycle checkpoints and DNA damage avoidance, cells have also evolved DNA damage tolerance mechanism, by which lesion-targeted mutation might occur in the genome during replication by specific DNA polymerases to bypass the lesions (translesion DNA synthesis, TLS), or mutation on undamaged DNA templates (untargeted mutation) might be induced. DNA polymerase zeta (pol zeta), which was found firstly in budding yeast Saccharomyces cerevisiae and consists of catalytic subunit scRev3 and stimulating subunit scRev7, has received more attention in recent years. Pol zeta is a member of DNA polymerase eta subfamily, which belongs to DNA polymerase B family, and exists in almost all eukaryotes. Human homolog of the scRev3 gene is located in chromosome region 6q21, and the mouse equivalent maps to chromosome 10, distal to the c-myb gene and close to the Macs gene. Alternative splicing, upstream out-of frame ATG can be found in yeast scRev3, mouse and human homologs. Furthermore, the sequence from 253-323 immediate upstream of the AUG initiator codon has the potential to form a stem-loop hairpin secondary structure in REV3 mRNA, suggesting that human REV3 protein may be expressed at low levels in human cells under normal growth conditions. The functional domain analysis showed that yeast Rev3-980 tyrosine in conserved region II is at the polymerase active site. Human REV3 amino acid residues 1 776-2 195 provide a REV7 binding domain, and REV7 amino acid residues 1-211 provide a bind domain for REV1, REV3 and REV7 itself. More interestingly, REV7 interacts with hMAD2 and therefore might function in the cell cycle control by affecting the activation of APC (anaphase promoting complex). Currently it has been known that pol zeta is involved in most spontaneous mutation, lesion-targeted mutation via TLS, chemical carcinogen induced untargeted mutation and somatic hypermutation of antibody genes in mammalian. In TLS pathway, pol zeta acts as a "mismatch extender" with combination of other DNA polymerases, such as pol iota. Unlike in yeast, it was found that pol zeta also functioned in mouse embryonic development more recently. It was hypothesized that the roles of pol zeta in TLS and cell cycle control might contribute to mouse embryonic lethality.
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PMID:DNA polymerase zeta: new insight into eukaryotic mutagenesis and mammalian embryonic development. 1280 Feb 16

In the budding yeast Saccharomyces cerevisiae, DNA polymerase zeta (pol zeta) is responsible for the great majority of mutations generated during error-prone translesion replication of DNA that contains UV-induced lesions. The catalytic subunit of pol zeta is encoded by the Rev3 gene. The orthologue of Rev3 has been cloned from higher eukaryotic cells, including human, but its role in mutagenesis and carcinogenesis remains obscure. Investigation into the cellular function of pol zeta has been hindered by the fact that Rev3 knockout mice do not survive beyond midgestation, and embryonic stem cells used to derive these mice are not genetically stable. We have generated a transgenic mouse that expresses antisense RNA transcripts to mRev3 endogeneous RNA. These mice are viable, have greatly reduced levels of Rev3 transcript, and have reduced levels of B cells and impaired development of high-affinity memory B cells. Here, we report that exposure of fibroblasts derived from these mice to UV resulted in a 4-5-fold reduction in mutant frequency at the hprt locus at every dose examined, and the mutation spectrum was highly aberrant compared with the control cells. In the control cells, 80% of the mutations were transitions and approximately 75% of these arose from photoproducts in the putative leading strand template. Strikingly, in transgenic cells, most of the mutations were transversions and there was a complete loss of strand bias. This mutation spectrum is highly aberrant and is similar to that induced by UV in human xeroderma pigmentosum variant cells, which lack polymerase eta. These data indicate that most UV-induced mutations are dependent on DNA pol zeta, a function that has been conserved from yeast to higher eukaryotic cells. However, in mammalian cells, other DNA polymerase(s) may accomplish error-prone translesion replication and are responsible for residual UV mutagenesis observed in the absence of pol zeta. Further, these data support a central role for DNA polymerase eta in the error-free bypass of UV photoproducts. The antisense Rev3 mice should be a useful model to study mutagenic lesion bypass by pol zeta in mammalian cells and to investigate the role this polymerase plays in carcinogenesis.
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PMID:Decreased frequency and highly aberrant spectrum of ultraviolet-induced mutations in the hprt gene of mouse fibroblasts expressing antisense RNA to DNA polymerase zeta. 1451 46

Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol zeta (DNA polymerase zeta) from Drosophila melanogaster (Dmpol zeta). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol zeta was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2',3'-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)(10:1) as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol zeta showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol zeta were not detected. Drosophila REV7 interacted with Dmpol zeta in vitro, but did not influence the DNA synthesis activity of Dmpol zeta. The present study is the first report about characterization of purified pol zeta from multicellular organisms, and the second concerning the characterization of yeast pol zeta.
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PMID:Purification of Drosophila DNA polymerase zeta by REV1 protein-affinity chromatography. 1517 13

Rev3L encodes the catalytic subunit of DNA polymerase zeta (pol zeta) in mammalian cells. In yeast, pol zeta helps cells bypass sites of DNA damage that can block replication enzymes. Targeted disruption of the mouse Rev3L gene causes lethality midway through embryonic gestation, and Rev3L-/- mouse embryonic fibroblasts (MEFs) remain in a quiescent state in culture. This suggests that pol zeta may be necessary for tolerance of endogenous DNA damage during normal cell growth. We report the generation of mitotically active Rev3L-/- MEFs on a p53-/- genetic background. Rev3L null MEFs exhibited striking chromosomal instability, with a large increase in translocation frequency. Many complex genetic aberrations were found only in Rev3L null cells. Rev3L null cells had increased chromosome numbers, most commonly near pentaploid, and double minute chromosomes were frequently found. This chromosomal instability associated with loss of a DNA polymerase activity in mammalian cells is similar to the instability associated with loss of homologous recombination capacity. Rev3L null MEFs were also moderately sensitive to mitomycin C, methyl methanesulfonate, and UV and gamma-radiation, indicating that mammalian pol zeta helps cells tolerate diverse types of DNA damage. The increased occurrence of chromosomal translocations in Rev3L-/- MEFs suggests that loss of Rev3L expression could contribute to genome instability during neoplastic transformation and progression.
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PMID:Loss of DNA polymerase zeta causes chromosomal instability in mammalian cells. 1639 25

The Rev1 protein lies at the root of mutagenesis in eukaryotes. Together with DNA polymerase zeta (Rev3/7), Rev1 function is required for the active introduction of the majority of mutations into the genomes of eukaryotes from yeast to humans. Rev1 and polymerase zeta are error-prone translesion DNA polymerases, but Rev1's DNA polymerase catalytic activity is not essential for mutagenesis. Rather, Rev1 is thought to contribute to mutagenesis principally by engaging in crucial protein-protein interactions that regulate the access of translesion DNA polymerases to the primer terminus. This inference is based on the requirement of the N-terminal BRCT (BRCA1 C-terminal) domain of Saccharomyces cerevisiae Rev1 for mutagenesis and the interaction of the C-terminal region of mammalian Rev1 with several other translesion DNA polymerases. Here, we report that S. cerevisiae Rev1 is subject to pronounced cell cycle control in which the levels of Rev1 protein are approximately 50-fold higher in G(2) and throughout mitosis than during G(1) and much of S phase. Differential survival of a rev1Delta strain after UV irradiation at various points in the cell cycle indicates that this unanticipated regulation is physiologically relevant. This unexpected finding has important implications for the regulation of mutagenesis and challenges current models of error-prone lesion bypass as a process involving polymerase switching that operates mainly during S phase to rescue stalled replication forks.
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PMID:The critical mutagenic translesion DNA polymerase Rev1 is highly expressed during G(2)/M phase rather than S phase. 1675 Dec 78

Exposure to ultraviolet light induces a number of forms of damage in DNA, of which (6-4) photoproducts present the most formidable challenge to DNA replication. No single DNA polymerase has been shown to bypass these lesions efficiently in vitro suggesting that the coordinate use of a number of different enzymes is required in vivo. To further understand the mechanisms and control of lesion bypass in vivo, we have devised a plasmid-based system to study the replication of site-specific T-T(6-4) photoproducts in chicken DT40 cells. We show that DNA polymerase zeta is absolutely required for translesion synthesis (TLS) of this lesion, while loss of DNA polymerase eta has no detectable effect. We also show that either the polymerase-binding domain of REV1 or ubiquitinated PCNA is required for the recruitment of Polzeta as the catalytic TLS polymerase. Finally, we demonstrate a previously unappreciated role for REV1 in ensuring bypass synthesis remains in frame with the template. Our data therefore suggest that REV1 not only helps to coordinate the delivery of DNA polymerase zeta to a stalled primer terminus but also restrains its activity to ensure that nucleotides are incorporated in register with the template strand.
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PMID:REV1 restrains DNA polymerase zeta to ensure frame fidelity during translesion synthesis of UV photoproducts in vivo. 1895 31

DNA polymerase zeta (Polzeta) is an error-prone DNA polymerase involved in translesion DNA synthesis. Polzeta consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847-1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Polzeta to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.
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PMID:Crystal structure of human REV7 in complex with a human REV3 fragment and structural implication of the interaction between DNA polymerase zeta and REV1. 2016 94


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