Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The identification and purification of human cell proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40) origin is described. Using these proteins, complete SV40 DNA replication was reconstituted with only purified DNA replication factors: SV40 large tumor antigen (TAg), replication protein A (RPA), DNA topoisomerases I and II, DNA polymerase alpha-primase, replication factor C (RFC), the proliferating cell nuclear antigen (PCNA), DNA polymerase delta, maturation factor 1 (MF1), and DNA ligase I. MF1, a 5' to 3' exonuclease and DNA ligase I were both identified as essential components for production of covalently closed circular relaxed (form I) DNA. MF1 is probably the same exonuclease previously shown by others to function during DNA synthesis on artificial DNA templates or in conjunction with DNA polymerase alpha from the SV40 origin. Combined with these previous studies, our results suggest that MF1 functions to remove an RNA primer attached to every Okazaki fragment during lagging strand DNA synthesis. Interestingly, whereas mammalian DNA ligase I functioned in the reconstituted replication system, mammalian DNA ligase III did not substitute and the phage T4 DNA ligase functioned inefficiently, suggesting that DNA ligase I has a specific role as a replicative DNA ligase in eukaryotic cells.
 ...
PMID:Reconstitution of complete SV40 DNA replication with purified replication factors. 814 77

The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.
 ...
PMID:Further purification and characterization of a multienzyme complex for DNA synthesis in human cells. 830 Jul 57

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.
 ...
PMID:Further characterization of the human cell multiprotein DNA replication complex. 853 May 40

This study describes characteristics of a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC-2, which was established by repeated in vitro exposures of a 6-fold MMC-resistant variant (J82/MMC) to 18 nM MMC. A 9.6-fold higher concentration of MMC was required to kill 50% of the J82/MMC-2 sub-line compared with parental cells (J82/WT). NADPH cytochrome P450 reductase and DT-diaphorase activities were significantly lower in J82/MMC-2 cells compared with J82/WT, suggesting that reduced sensitivity of J82/MMC-2 cells to MMC resulted from impaired drug activation. Consistent with this hypothesis, the formation of MMC-alkylating metabolites was significantly lower in J82/MMC-2 cells compared with J82/WT. Furthermore, DT-diaphorase activity in J82/MMC-2 cells was significantly lower compared with the 6-fold MMC-resistant variant. Glutathione (GSH) levels were comparable in all 3 cell lines. Although GSH transferase (GST) activity was significantly higher in the J82/MMC-2 cells compared with J82/WT, this enzyme activity did not differ between 6- and 9.6-fold MMC-resistant variants. Whereas DNA polymerase alpha mRNA expression was comparable in these cell lines, levels of DNA ligase I mRNA were slightly lower in both MMC-resistant variants relative to J82/WT. However, the DNA polymerase beta mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J82/WT or J82/MMC. Thus, emergence of a higher level of resistance to MMC in J82/MMC-2 cells compared with J82/MMC may be attributed to (i) impaired drug activation through further reduction in DT-diaphorase activity and (ii) enhanced DNA repair through over-expression of DNA polymerase beta.
 ...
PMID:Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line. 863 3

Base excision repair (BER) is a cellular defense mechanism repairing modified bases in DNA. Recently, a G:U repair reaction has been reconstituted with several purified enzymes from Escherichia coli (Dianov, G., and Lindahl, T.(1994) Curr. Biol. 4, 1069-1076). Using bovine testis crude nuclear extract, we have shown that G:U is repaired efficiently in vitro, and DNA polymerase beta (beta-pol) is responsible for the single nucleotide gap-filling synthesis (Singhal, R. K., Prasad, R., and Wilson, S. H.(1995) J. Biol. Chem. 270, 949-957). To investigate potential interaction of beta-pol with other BER protein(s), we developed affinity chromatography matrices by cross-linking purified rat beta-pol or antibody against beta-pol to solid supports. Crude nuclear extract from bovine testis was applied to these affinity columns, which were then extensively washed. Proteins that bound specifically to the affinity columns were co-eluted in a complex with beta-pol. This complex had a molecular mass of approximately 180 kDa and was able to conduct the complete uracil-initiated BER reaction. The BER complex contained both beta-pol and DNA ligase I. An antibody to beta-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both beta-pol and DNA ligase I. Furthermore, DNA ligase I and beta-pol were co-immunoprecipitated from the testis nuclear extract with anti beta-pol IgG. Thus, we conclude that beta-pol and DNA ligase I are components of a multiprotein complex that performs BER.
 ...
PMID:Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis. 866 74

Repair of alkylated bases in DNA is performed by O6-methylguanine-DNA methyltransferase (MGMT) and a set of enzymes of the base excision repair pathway involving N-methylpurine-DNA glycosylase (MPG), apurinic endonuclease (APE), DNA polymerase beta (Pol beta) and DNA ligase. The level of expression of these enzymes may exert a profound effect on resistance of cells towards alkylating drugs. We have comparatively analyzed the expression of MGMT and the different base excision repair genes in rat hepatoma cells (line H4IIE) after exposure to alkylating agents, X-rays and the glucocorticoid hormone dexamethasone. Furthermore, the effect of these agents on the activity of the cloned human MGMT promoter was assayed. Exposure of cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ionizing radiation increased MGMT mRNA levels up to 4.5-fold. Under the same conditions of treatment, exerting only a weak toxic effect, MPG and DNA ligase I mRNA levels were not enhanced, whereas the amounts of APE and Pol beta mRNA transiently increased by approximately 2-fold after X-ray and MNNG treatment, respectively. Dexamethasone induced both MGMT, APE and Pol beta mRNA and the induction paralleled the increase in mRNA of the glucocorticoid-dependent gene tyrosine aminotransferase. The observed increase in MGMT mRNA was due to promoter activation, which was shown in transient transfection assays with MGMT promoter-CAT reporter constructs in H4IIE cells. In these assays, the human MGMT promoter was found to be induced by methylating agents (MNNG and methyl methanesulfonate), ionizing radiation and dexamethasone. Weak induction of the promoter was observed after UV irradiation. Treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate was ineffective in promoter activation. The transfected MGMT promoter was not inducible by mutagens in HeLa S3 cells, which do not respond with induction of the endogenous MGMT gene. This is the first report showing hormone induction of a DNA repair gene (MGMT). The induction of MGMT and other genes encoding enzymes involved in DNA alkylation damage repair may be relevant in cancer therapy by causing resistance of tumor cells to alkylating drugs.
 ...
PMID:Induction of the alkyltransferase (MGMT) gene by DNA damaging agents and the glucocorticoid dexamethasone and comparison with the response of base excision repair genes. 896 45

Base excision repair is a major mechanism for correcting aberrant DNA bases. We are using an in vitro base excision repair assay to fractionate and purify proteins from a human cell extract that are involved in this type of repair. Three fractions are required to reconstitute base excision repair synthesis using a uracil-containing DNA as a model substrate. We previously showed that one fraction corresponds to DNA polymerase beta. A second fraction was extensively purified and found to possess uracil-DNA glycosylase activity and was identified as the product of the UNG gene. A neutralizing antibody to the human UNG protein inhibited base excision repair in crude extract by at least 90%. The third fraction was highly purified and exhibited apurinic/apyrimidinic (AP) endonuclease activity. Immunoblot analysis identified HAP1 as the major polypeptide in fractions possessing DNA repair activity. Recombinant versions of UNG, HAP1, and DNA polymerase beta were able to substitute for the proteins purified from human cells. Addition of DNA ligase I led to ligated repair products. Thus, complete base excision repair of uracil-containing DNA was achieved by a combination of UNG, HAP1, DNA polymerase beta, and DNA ligase I. This is the first complete reconstitution of base excision repair using entirely eukaryotic proteins.
 ...
PMID:Reconstitution of human base excision repair with purified proteins. 920 Jul 7

The Herpesviridae comprise a large class of animal viruses of considerable public health importance. Of the Herpesviridae, replication of herpes simplex virustype-1 (HSV-1) has been the most extensively studied. The linear 152-kbp HSV-1 genome contains three origins of DNA replication and approximately 75 open-reading frames. Of these frames, seven encode proteins that are required for originspecific DNA replication. These proteins include a processive heterodimeric DNA polymerase, a single-strand DNA-binding protein, a heterotrimeric primosome with 5'-3' DNA helicase and primase activities, and an origin-binding protein with 3'-5' DNA helicase activity. HSV-1 also encodes a set of enzymes involved in nucleotide metabolism that are not required for viral replication in cultured cells. These enzymes include a deoxyuridine triphosphatase, a ribonucleotide reductase, a thymidine kinase, an alkaline endo-exonuclease, and a uracil-DNA glycosylase. Host enzymes, notably DNA polymerase alpha-primase, DNA ligase I, and topoisomerase II, are probably also required. Following circularization of the linear viral genome, DNA replication very likely proceeds in two phases: an initial phase of theta replication, initiated at one or more of the origins, followed by a rolling-circle mode of replication. The latter generates concatemers that are cleaved and packaged into infectious viral particles. The rolling-circle phase of HSV-1 DNA replication has been reconstituted in vitro by a complex containing several of the HSV-1 encoded DNA replication enzymes. Reconstitution of the theta phase has thus far eluded workers in the field and remains a challenge for the future.
 ...
PMID:Herpes simplex virus DNA replication. 924 11

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.
 ...
PMID:Identification of DNA replication and cell cycle proteins that interact with PCNA. 939 13

We previously identified and characterized the human leukemia (HL-60) cell DNA synthetic machinery as a multiprotein form of DNA polymerase, which was designated the DNA synthesome. This multiprotein replication complex contains DNA polymerases alpha and delta, primase, replication factor C, replication protein A, helicase, poly(ADPribose) polymerase, proliferating cell nuclear antigen, DNA ligase I, and topoisomerases I and II. Recently, the HeLa cell-derived DNA synthesome was identified as a discrete high molecular weight protein band in native polyacrylamide gels. Here, we report our findings regarding the change in the organizational status of the DNA synthesome when HL-60 cells undergo either terminal differentiation or temporary G1 growth arrest. We observed that the HL-60 cell DNA synthesome also migrates as a discrete high molecular weight protein band in nondenaturing polyacrylamide gels. This high molecular weight protein band was present in nuclei derived from both actively cycling cells and aphidicolin-arrested cells but was absent in TPA-induced terminally differentiated cells. We also found that DNA polymerase delta, replication factor C, and proliferating cell nuclear antigen are absent in cells that are induced to differentiate in response to 12-O-tetradecanoyl phorbol-13-acetate treatment but are present in actively cycling cells. The level of replication protein A in differentiated cells was similar to that of cycling cells, whereas the level of annexin I, a cytoskeleton protein, is higher in differentiated cells than it is in actively cycling cells. We conclude that the DNA synthesome remains integrated and inactive in temporarily growth-arrested cells but is disassembled in differentiated cells. Furthermore, we conclude that disassembly of the organized replication complex is a specific cellular event in the process of permanent cell cycle exit and that the process leading to disassembly may be regulated, in part, at the level of gene transcription.
 ...
PMID:The biochemical status of the DNA synthesome can distinguish between permanent and temporary cell growth arrest. 941 24


<< Previous 1 2 3 4 5 6 7 8 Next >>