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)

Transgenic systems, both cell lines and mice with gain or loss of function, are being used in order to modulate the expression of DNA repair proteins, thus allowing to assess their contribution to the defense against genotoxic mutagens and carcinogens. In this review, questions have been addressed concerning the use of transgenic systems in elucidating critical primary DNA lesions, their conversion into genotoxic endpoints, low-dose effects, and the relative contribution of individual cellular functions in defense. It has been shown that the repair protein alkyltransferase (MGMT) is decisive for protection against methylating and chloroethylating compounds. Protection pertains also to tumor formation, as revealed by the response of MGMT transgenic and knockout mice. Overexpression of genes involved in base excision repair (N-methylpurine-DNA glycosylase, apurinic endonuclease, DNA polymerase beta) is in most cases not beneficial in increasing the protection level, whereas their down-modulation or inactivation increases cellular sensitivity. This indicates that non-repaired base N-alkylation lesions and/or repair intermediates possess genotoxic potential. Modulation of mismatch repair and poly(ADP)ribosyl transferase has also been shown to affect the cellular response to alkylating agents. Furthermore, the role of Fos, Jun and p53 in cellular defense against alkylating mutagens is discussed.
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PMID:Transgenic systems in studies on genotoxicity of alkylating agents: critical lesions, thresholds and defense mechanisms. 974 64

The products of the tumor suppressor genes are considered to function as specific inhibitors of tumor cell growth. In this communication, we present evidence to show that these proteins inhibit tumor cell proliferation by participating in the activation of tumor cell differentiation. The ML-1 human myeloblastic leukemia cells used in this study proliferate when treated with insulin-like growth factor I and transferrin but differentiate to monocytes when exposed to tumor necrosis factor alpha or transforming growth factor beta1, or to macrophage-like cells when treated with both these cytokines. Initiation of proliferation but not of differentiation was followed by a 20- to 25-fold increase in the nuclear level of the DNA polymerase-associated processivity factor PCNA and of the proliferation-specific transcription factor E2F1. In contrast, induction of differentiation but not of proliferation was followed by a 25- to 30-fold increase in the nuclear level of the tumor suppressor proteins p53 (wild type), pRb, and p130/Rb2 and of the p53-dependent cyclin kinase inhibitor p21/Cip1. p53 and p21/Cip1, respectively, inhibit the expression and activation of PCNA, whereas p130 and pRb, respectively, inhibit the expression and activation of E2F1. As a result, G1-S-associated DNA and mRNA synthesis is inhibited, growth uncoupled from differentiation, and maturation enabled to proceed. Where this function of the tumor suppressor proteins is impaired, the capacity for differentiation is lost, which leads to the sustained proliferation that is characteristic of the cancer cell.
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PMID:Tumor suppressor proteins as regulators of cell differentiation. 976 53

Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumor suppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase alpha-primase complex (pol-prim), and the cellular single-strand DNA binding protein RPA. Highly purified p53 protein bound to immobilized T-ag with an apparent binding constant of 2 x 10(8) M(-1). Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4 x 10(8) M(-1), when RPA was coupled to the sensor chip via its smallest subunit, and 1 x 10(8) M(-1), when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase alpha-primase complex (pol-prim) with a K(A) value of 1 x 10(10) m(-1). Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2 x 10(10) m1(-1) and 5 X 10(9) M(-1), respectively. Complex formation was also observed with a p180/p68 heterodimer, and again with a binding constant similar. Hence, there was no synergistic effect when p53 bound to higher order complexes of pol-prim. A truncated form of p53, consisting of amino acids 1-320, bound pol-prim by four orders of magnitude less efficiently. Therefore, an intact C-terminus of p53 seems to be important for efficient binding to pol-prim. It was also tried to measure complex formation between p53, pol-prim, and T-ag. However there was no evidence for the existence of a ternary complex consisting of T-ag, pol-prim, and p53.
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PMID:Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase alpha. 998 27

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) is a nucleoside phosphonate analog which in its active diphosphorylated form is known to inhibit herpesvirus DNA polymerase. In this study, we have demonstrated that, in a dose-dependent manner, this compound irreversibly suppressed proliferation of cells infected with human papillomavirus (HPV), which does not possess a viral DNA polymerase. To elucidate the mechanism of cell growth inhibition, cell cycle indicator-regulator expression, thymidine incorporation, transcript levels of apoptosis factors, and anabolic products of HPMPC following drug treatment were evaluated. HPMPC treatment reduced WAF1 (p21) levels independent of those of p53, while proliferating cell nuclear antigen increased. However, in comparison to controls, HPMPC-treated cells displayed a decrease in thymidine incorporation, indicating an inhibition of host DNA polymerase activity. In normal primary keratinocytes, HPMPC predominantly accumulated in the form of the choline adduct HPMPCp-choline. However, in HPV type 16-transformed keratinocytes, HPMPCpp was the most abundant anabolic product, with little HPMPCp-choline having formed. The data imply that an unrecognized viral factor is modulating the conversion of nucleotides, including HPMPC, to the triphosphorylated form.
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PMID:Selective inhibition of human papillomavirus-induced cell proliferation by (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine. 1022 36

The Epstein-Barr Virus (EBV) immediate-early protein BRLF1 is one of two transactivators which mediate the switch from latent to lytic replication in EBV-infected cells. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of BRLF1 on cell cycle progression. A replication-deficient adenovirus expressing BRLF1 (AdBRLF1) was used to infect normal human fibroblasts and various epithelial cell lines. BRLF1 expression induced S phase entry in contact-inhibited fibroblasts and in the human osteosarcoma cell line U-2 OS. AdBRLF1 infection produced a dramatic increase in the level of E2F1 but not E2F4. In contrast, the levels of Rb, p107, and p130 were decreased in AdBRLF1-infected cells. Electrophoretic mobility shift assays confirmed an increased level of free E2F1 in the AdBRLF1-infected human fibroblasts. Consistent with the previously described effect of E2F1, AdBRLF1-infected fibroblasts had increased levels of p53 and p21 and died by apoptosis. BRLF1-induced activation of E2F1 may be required for efficient EBV lytic replication, since at least one critical viral replication gene (the viral DNA polymerase) is activated by E2F (C. Liu, N. D. Sista, and J. S. Pagano, J. Virol. 70:2545-2555, 1996).
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PMID:The Epstein-Barr virus protein BRLF1 activates S phase entry through E2F1 induction. 1040 Jul 50

Clarification of somatic mutations during the progression of human astrocytomas is important in order to understand the mechanisms underlying the development of these tumors. We analyzed surgical specimens of human astrocytomas for mutations in the p53 gene using single-strand conformation polymorphism analysis of polymerase chain reaction product (PCR-SSCP analysis) at a low pH. Klenow fragment treatment after PCR amplification was an effective means to get rid of some extra bands on the SSCP gel. Five mutations in three of 24 astrocytomas were identified by this improved SSCP method. The frequency of p53 gene mutations in astrocytomas examined was 12.5%. Further examination by direct sequencing showed that all five mutants had single-base substitutions resulting in missense mutations. The present studies revealed a loss of heterozygosity and two point mutations on the remaining allele in one of the fibrillary astrocytomas. Finally, the improvement of PCR-SSCP analysis using Klenow treatment and low pH showed a distinct electrophoresis gel pattern and could be relevant for the prognosis of human astrocytomas.
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PMID:Distinct pattern of PCR-SSCP analysis of p53 mutations in human astrocytomas. 1045 62

Human Replication Protein A (hsRPA) is required for multiple cellular processes in DNA metabolism including DNA repair, replication and recombination. It binds single-stranded DNA with high affinity and interacts specifically with multiple proteins. hsRPA forms a heterotrimeric complex composed of 70-, 32- and 14-kDa subunits (henceforth RPA70, RPA32, and RPA14). The N-terminal 168 residues of RPA70 form a structurally distinct domain that stimulates DNA polymerase alpha activity, interacts with several transcriptional activators including tumor suppressor p53, and during the cell cycle it signals escape from the DNA damage induced G2/M checkpoint. We have solved the global fold of the fragment corresponding to this domain (RPA70 delta 169) and we find residues 8-108 of the N-terminal domain are structured. The remaining C-terminal residues are unstructured and may form a flexible linker to the DNA-binding domain of RPA70. The globular region forms a five-stranded anti-parallel beta-barrel. The ends of the barrel are capped by short helices. Two loops on one side of the barrel form a large basic cleft which is a likely site for binding the acidic motifs of transcriptional activators. Many lethal or conditional lethal yeast point mutants map to this cleft, whereas no mutations with severe phenotype have been found in the linker region.
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PMID:Human replication protein A: global fold of the N-terminal RPA-70 domain reveals a basic cleft and flexible C-terminal linker. 1052 7

Cells that have been irradiated with ultraviolet light (UV) suffer damage to their DNA, primarily in the form of covalent linkage between adjacent pyrimidines. Such photoproducts represent blocks to RNA and DNA polymerases and are potentially mutagenic. Blockage of RNA polymerase II by a photoproduct in the transcribed strand of an active gene leads to induction of the p53 protein, which induces pleiotropic responses that may include apoptotic cell death. If a cell survives, the blocked polymerase targets the nucleotide excision repair machinery to the site of the lesion, which is repaired in an error-free manner. Repair coupled to transcription in this manner strongly influences the mutation spectrum induced by UV, reducing the proportion of base substitutions that arise from photoproducts on the transcribed strand. If the damage persists when the DNA is replicated in S-phase, either because the cell is unable to repair the damage or because there is insufficient time between the induction of damage and the onset of S-phase. To do so, the replicative DNA polymerase complex may be blocked. In this situation, lesion bypass can be accomplished using an error-free mechanism, or using an error-prone mechanism that involves the newly described, non-processive DNA polymerase zeta encoded by the human homolog of the yeast REV3 gene.
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PMID:DNA repair, DNA replication, and UV mutagenesis. 1053 99

The telomere DNA polymerase (telomerase) and the tumor suppressor protein p53 are frequently associated with human cancers, and activation of telomerase and inactivation of p53 involved in cancer cell immortalization. In this report, we demonstrate a direct interaction of telomerase with p53 in the nuclear lysates of human breast cancer cells, and with recombinant human p53, by affinity chromatography and immunoprecipitation. On activity criteria, the interaction is between the carboxyl-terminal region of p53 and a region close to the amino-terminus of human telomerase-associated protein 1 (hTEP1). Incubation of recombinant p53 with nuclear telomerase extracts results in inhibition of telomerase activity, with the C-terminal region of p53 being essential for inhibition. This effect is not mediated by binding to telomerase substrate DNA, but requires the region near the N-terminus of hTEP1, in that a synthetic peptide derived from this region of hTEP1 similarly inhibits telomerase activity. Together, these in vitro interactions between telomerase and p53 suggest that the activity of telomerase may be regulated by p53, down-regulation of which in turn would favor up-regulation of telomerase activity in cancer cell development.
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PMID:Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro. 1059 87

Chloroacetaldehyde (CAA) reacts with DNA bases, forming hydroxyethano derivatives of different stability, which are subsequently converted into etheno (epsilon) adducts: epsilon A, epsilon C, epsilon G. DNA polymerase fingerprint analysis was used to study the distribution of CAA-induced modifications in the p53 sequence. A plasmid bearing cDNA containing the human p53 gene was reacted in vitro with CAA, then dehydrated for conversion of hydroxyethano into etheno adducts, and primer extension by T7 DNA polymerase in the presence of four dNTPs was performed. The DNA repair enzymes methylpurine-DNA glycosylase and Escherichia coli exonuclease III were used to convert epsilon A residues in the template into DNA strand breaks, which enabled precise localization of the epsilon A residues within the p53 gene. Hydroxyethano derivatives of adenine and cytosine in a template blocked T7 DNA polymerase and caused premature chain termination opposite adenine or one base before cytosine. After dehydration, both epsilon A and epsilon C were much more easily by-passed by T7 DNA polymerase. Formation of epsilon G was identified as 'stop bands' one base before guanine residues. Modification of cytosine and guanine was additionally recognized by weakening or disappearance of non-specific stops on an undamaged template, probably due to steric hindrance by the tertiary DNA structure for polymerase. Etheno adduction of cytosine and guanine relaxed the compact DNA structure and enabled DNA polymerase to by-pass. In exons 5-8 of p53, 143 out of 500 sites appeared to be damaged by CAA, with four particularly densely modified regions between codons 135-147, 218-222, 234-255 and 284-292. The pattern of modification followed the pattern of p53 mutations found in vinyl chloride-associated liver angiosarcomas in humans and rats, but only in regions that showed 100% homology with the human sequence. The factors that influence DNA damage and induction of mutations in the p53 gene by CAA and vinyl chloride are discussed.
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PMID:Localization of chloroacetaldehyde-induced DNA damage in human p53 gene by DNA polymerase fingerprint analysis. 1062 28


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