Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase δ

Yang, Chun li; Chang, Long Sheng; Zhang, Peng; Hao, Huiling; Zhu, Liang; Toomey, Ngoc L.; Lee, Marietta Y.W.T.

doi:10.1093/nar/20.4.735

Cited by 73 publications

(55 citation statements)

References 71 publications

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“…After heating at 95°C for 5 min and chilling on ice, products were separated on 6% PAGE in Tris borate-EDTA with 7 M urea. (21). The resequencing of a number of partial cDNA clones that had been generated by PCR amplification of poly(A) ϩ RNA from HepG2 cells using primers HGF3/HR3 and H3/HGR5 (18) revealed that codons for Arg-30 and Tyr-472 were present in some clones and were likely correct, whereas codons for His-119, Asn-173, and Gly-776 were not present in any of our clones.…”

Section: Sds-polyacrylamide Gel Electrophoresis and Proteinmentioning

confidence: 77%

Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

Zhou¹,

Tan

et al. 1996

Journal of Biological Chemistry

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The catalytic subunit of human DNA polymerase ␦ has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a M r ‫؍‬ ϳ125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase ␦. The recombinant protein was purified to near homogeneity (approximately 1200-fold) from insect cells by chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3 -5 exonuclease activities. The properties of the recombinant catalytic subunit were compared with those of the native heterodimeric DNA polymerase ␦ isolated from fetal calf thymus, and the enzymes were found to differ in several respects. Although the native heterodimer is equally active with either Mn 2؉ or Mg 2؉ as divalent cation activator, the recombinant catalytic subunit is approximately 5-fold more active in Mn 2؉ than in Mg 2؉ . The most striking difference between the two proteins is the response to the proliferating cell nuclear antigen (PCNA). The activity and processivity of native DNA polymerase ␦ are markedly stimulated by PCNA whereas it has no effect on the recombinant catalytic subunit. These results suggest that the small subunit of DNA polymerase ␦ is essential for functional interaction with PCNA.Although genetic studies in yeast have suggested that three distinct DNA polymerases (␣, ␦, and ⑀) are required for nuclear DNA replication in eukaryotes (1-4), only two DNA polymerases (␣ and ␦) were found to be essential components of an in vitro reconstituted SV40 DNA replication system (5); pol 1 ␣, with its tightly associated primase activity, was shown to be required for the synthesis of primers for both leading and lagging strands, whereas pol ␦ and its accessory proteins, proliferating cell nuclear antigen (PCNA) and replication factor C, also known as activator 1, were found to be required for leading strand synthesis and for completing Okazaki fragments initiated by pol ␣/primase. More recently, elucidation of the roles of DNA polymerases ␣, ␦, and ⑀ in DNA replication was approached by UV cross-linking of polymerases to nascent DNA within replicating chromosomes (6). These studies showed that only pol ␣ and pol ␦ were photolabeled by nascent SV40 DNA whereas pol ␣, pol ␦, and pol ⑀ were all photolabeled by nascent cellular DNA, suggesting that the replication of SV40 chromosomes may require only a subset of the proteins required for replication of cellular chromosomes. Thus, the roles of pol ␦ and pol ⑀ in cellular DNA replication may not be resolved by studies of the replication of DNA viruses; rather, studies of the properties of the replicative DNA polymerases and their interactions with other replication proteins will be necessary to answer these questions.pol ␦ has been purified from a number of eukaryotic sources including calf thymus (7), budding yeast (8), mouse cells (9), and Drosophila melanogaster (10). The enzyme is usually isol...

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Section: Sds-polyacrylamide Gel Electrophoresis and Proteinmentioning

confidence: 77%

Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

Zhou¹,

Tan

et al. 1996

Journal of Biological Chemistry

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“…Arginine residues at codon 506 of the POLD1 gene are highly conserved from yeast to mammals 21,44 and, therefore, R506H in DLD-1 cells is regarded as impairing the protein function and, consequently, as a dysfunctional mutation. 44 Valine residues at codon 312 are also conserved between mouse and human, although differing in yeast.…”

Section: Resultsmentioning

confidence: 99%

“…As pol epsilon possesses a relatively high processivity, it is now widely accepted that in eukaryotic cells pol delta and epsilon are responsible for the lagging-and leading-strand synthesis, respectively, 19 although pol epsilon has been demonstrated not to be essential in yeast. 20 The catalytic subunits of pol delta and epsilon, for example, p125 [21][22][23] and p261 24,25 in mammals, include the 3¢ exonuclease, that is, proofreading, domains. Indeed, in the yeast mutants in which pol delta or epsilon proofreading is selectively inactivated, the mutations rates are 10-100 times higher than the wild-type strains.…”

Section: Introductionmentioning

confidence: 99%

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

et al. 2010

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Genomic sequences encoding the 3¢ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms. Keywords: polymerase delta; polymerase epsilon; proofreading domain; colorectal cancer INTRODUCTION Mutation rates on the genome are invariably regulated and typically suppressed to an extremely low level, such as 10 À10 per base replicated, in the organisms. 1 The high fidelity of DNA replication is achieved by several molecular mechanisms. The frequency of erroneous nucleotide incorporation is indeed extremely low compared with that expected from base-pairing energetics, and the vast reduction of the misincorporation frequency involves three steps of molecular events: (a) correct incorporation of complementary nucleotides by DNA polymerases, (b) removal of the newly added nucleotides, particularly incorrectly paired nucleotides, by the 3¢ exonuclease activities associated with the DNA polymerases (proofreading) and (c) postreplicative scanning of mispaired bases by DNA mismatch repair (MMR). 2,3 Various Escherichia coli (E. coli) mutator strains have been used to study these molecular mechanisms by which organisms maintain their mutation rates at very low levels. The dnaQ + (mutD + ) gene of E. coli encodes the epsilon subunit of the DNA polymerase III holoenzyme that is involved in 3¢ exonuclease proofreading activity, 4 and, therefore, the mutation frequencies in the dnaQ mutators are sometimes 1000-10 000 times higher than the wild-type levels. 4,5 In E. coli, MMR is chiefly directed by the proteins encoded by the three genes: mutS + , mutL + and mutH + . 6 The mutS or mutL mutators exhibit an approximately 100 times increase in the mutation frequency. 7 Thus, polymerase proofreading and MMR are the two major

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“…Human Pol d4 is a heterotetramer of p125, p50, p68, and p12 subunits, like yPold4 in S. pombe. The catalytic core region of human p125 was found to share >60% similarities with those of S. cerevisiae and S. pombe Pol d, indicating a strong evolutionary conservation Yang et al, 1992]. However, p68 and p12 are poorly conserved with their yeast counterparts .…”

Section: Introductionmentioning

confidence: 99%

“…A second form of ''Pol d'' with a larger catalytic subunit was discovered and is now known as Pol e [Syvaoja et al, 1990;Pospiech and Syvaoja, 2003;Pursell and Kunkel, 2008]. Molecular cloning of p125 showed that the catalytic core was highly conserved in evolution from T4 bacteriophage to human Hao et al, 1992;Yang et al, 1992], and Pol d is now classified as a member of the B family polymerases. Closely allied with the early history of Pol d are studies of the eukaryotic DNA-sliding clamp, proliferating cell nuclear antigen (PCNA; [Kuriyan and O'Donnell, 1993;Moldovan et al, 2010]), which was discovered as a processivity factor that stimulated Pol d activity [Prelich et al, 1987].…”

Section: Introductionmentioning

confidence: 99%

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

Lee

Zhang

Lin

et al. 2012

Environ and Mol Mutagen

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The p12 subunit of polymerase delta (Pol d) is degraded in response to DNA damage induced by UV, alkylating agents, oxidative, and replication stresses. This leads to the conversion of the Pol d4 holoenzyme to the heterotrimer, Pol d3. We review studies that establish that Pol d3 formation is an event that could have a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair. p12 degradation is dependent on the apical ataxia telangiectasia and Rad3 related (ATR) kinase and is mediated by the ubiquitin-proteasome system. Pol d3 exhibits properties of an ''antimutator'' polymerase, suggesting that it could contribute to an increased surveillance against mutagenesis, for example, when Pol d carries out bypass synthesis past small base lesions that engage in spurious base pairing. Chromatin immunoprecipitation analysis and examination of the spatiotemporal recruitment of Pol d to sites of DNA damage show that Pol d3 is the primary form of Pol d associated with cyclobutane pyrimidine dimer lesions and therefore should be considered as the operative form of Pol d engaged in DNA repair. We propose a model for the switching of Pol d with translesion polymerases, incorporating the salient features of the recently determined structure of monoubiquitinated proliferating cell nuclear antigen and emphasizing the role of Pol d3. Because of the critical role of Pol d activity in DNA replication and repair, the formation of Pol d3 in response to DNA damage opens the prospect that pleiotropic effects may ensue. This opens the horizons for future exploration of how this novel response to DNA damage contributes to genomic stability. Environ. Mol. Mutagen. 53:683-698, 2012. V V C 2012 Wiley Periodicals, Inc.

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Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase δ

Cited by 73 publications

References 71 publications

Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

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