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)

Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polymerase (E9), an essential and stoichiometric component of the processive polymerase (A20), a single-strand DNA-binding protein (I3), a type I topoisomerase (H6), the uracil DNA glycosylase (D4), a nucleic acid-independent nucleoside triphosphatase (D5), a serine/threonine protein kinase (B1), and a Holliday Junction resolvase (A22). All of these factors work in concert to faithfully duplicate the viral genome. Although a replication origin has not been defined for the poxviruses, cis-acting sequences found within the telomeric 200 bp have been implicated as necessary and sufficient for minichromosome replication. Replication occurs within cytoplasmic foci from approx 3 to 12 h postinfection. This chapter includes several methodologies to assay and quantitate replication in vivo, visualize replication foci microscopically, and test the integrity of central replication enzymes in vitro.
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PMID:Methods for analysis of poxvirus DNA replication. 1511 16

The mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase beta (beta-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce beta-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, beta-pol-haploinsufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young beta-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and beta-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for beta-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases.
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PMID:Imbalanced base excision repair in response to folate deficiency is accelerated by polymerase beta haploinsufficiency. 1521 23

Human herpesvirus 6 (HHV-6) is latent in the majority of the adult population. Due to its ability of causing opportunistic infections, alone or in concert with the other beta-herpesviruses human cytomegalovirus (HCMV) and HHV-7, its importance as a pathogen in immunocompromised patients has increasingly been recognized. We here report the characterization of the main antiviral target, the HHV-6 DNA polymerase, expressed in a eukaryotic in vitro transcription/translation assay. This technique represents a fast and straightforward approach for the study of existing and new inhibitors of HHV-6 DNA polymerase. The present study shows that ganciclovir is less active against HHV-6, as compared to its activity against HCMV, both in cell culture and at the enzymatic (i.e. DNA polymerase) level. Recently, a mutant HHV-6 strain carrying an amino acid substitution in the ganciclovir phosphorylating pU69 kinase has been isolated both from patients and in cell culture. The strain isolated in vitro, however, carried an additional mutation in the viral DNA polymerase. From our experiments presented here, we conclude that the pU69 M(318)V amino acid substitution rather than the A(961)V substitution in HHV-6 DNA polymerase, is responsible for the ganciclovir-resistant phenotype.
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PMID:Human herpesvirus 6 DNA polymerase: enzymatic parameters, sensitivity to ganciclovir and determination of the role of the A961V mutation in HHV-6 ganciclovir resistance. 1545 Nov 75

Deoxycytidine kinase (dCK) plays a central role in the deoxynucleoside salvage processes, phosphorylating dC, dA, and dG to their monophosphates. In mammalian cells, the major source of dTTP comes also from dC via dCMP deaminase. Moreover, based on its broad substrate specificity, this enzyme is responsible for the activation of several nucleoside analogues of therapeutical importance, influencing the sensitivity of malignant tissues towards chemotherapy. The expression of dCK is highest in different lymphoid cells/tissues, in embryonic cells and in most malignant cells (2, 7, 13-15, 18). The activity of dCK is not cell cycle-regulated. In contrast to this, dCK activity was found to be elevated several fold upon short-term treatments of normal human lymphocytes with therapeutic nucleoside analogs, and other genotoxic agents as well as by DNA damaging agents including the DNA polymerase inhibitor aphidicolin, the topoisomerase II inhibitor etoposide and gamma-irradiation, which might be a potentially important phenomenon with respect to the clinical practice, too. These findings indicated that the main trigger of activation could be the damaged DNA itself, and the biological relevance might be to supply the dNTPs for the enhanced DNA repair. Activation of dCK was paralleled by elevated levels of intracellular dATP, raising the possibility that dCK activation is linked to the induction of apoptosis. With regard to the mechanism of enzyme activation, no changes were found in the protein and mRNA levels of dCK upon stimulation, while the activation process was calcium dependent and comprised a protein phosphorylation step. A positive correlation was found between the enzymatic activity and the native immunoreactivity of dCK, strongly arguing that dCK undergoes a conformational change during activation, which results in the formation of a catalytically more active steric structure (8-11, 22, 26, 32-34, 35, 36).
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PMID:[Special function of deoxycytidine kinase (dCK) in the activation of chemotherapeutic nucleoside analogs and in the inhibition of cell proliferation]. 1552 Aug 73

Recently, we developed an in vitro system using human uracil DNA glycosylase (UDG), AP endonuclease (APE), DNA polymerase beta (pol beta) and rotationally positioned DNA containing a single uracil associated with a 'designed' nucleosome, to test short-patch base excision repair (BER) in chromatin. We found that UDG and APE carry out their catalytic activities with reduced efficiency on nucleosome substrates, showing a distinction between uracil facing 'out' or 'in' from the histone surface, while DNA polymerase beta (pol beta) is completely inhibited by nucleosome formation. In this report, we tested the inhibition of BER enzymes by the N-terminal 'tails' of core histones that take part in both inter- and intra-nucleosome interactions, and contain sites of post-translational modifications. Histone tails were removed by limited trypsin digestion of 'donor' nucleosome core particles and histone octamers were exchanged onto a nucleosome-positioning DNA sequence containing a single G:U mismatch. The data indicate that UDG and APE activities are not significantly enhanced with tailless nucleosomes, and the distinction between rotational settings of uracil on the histone surface is unaffected. More importantly, the inhibition of pol beta activity is not relieved by removal of the histone tails, even though these tails interact with DNA in the G:U mismatch region. Finally, inclusion of X-ray cross complement group protein 1 (XRCC1) or Werner syndrome protein (WRN) had no effect on the BER reactions. Thus, additional activities may be required in cells for efficient BER of at least some structural domains in chromatin.
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PMID:Base excision repair in nucleosomes lacking histone tails. 1559 Mar 28

Base excision repair (BER) averts the cytotoxic and mutagenic effects of most endogenously produced DNA damage, including lesions that arise spontaneously due to the intrinsic instability of DNA or modifications that are formed from reactions with intracellular chemicals, such as reactive oxygen species and alkylating agents. Defects in the BER process have been associated with cancer susceptibility and neurodegenerative disorders. In its most simplistic form, BER can be fully reconstituted with a minimum of four human proteins and is completed in just five sequential steps: (i) excision of an inappropriate base by a DNA glycosylase (e.g., uracil DNA glycosylase); (ii) incision of the DNA backbone immediately adjacent to the resulting abasic site by apurinic/apyrimidimic endonuclease 1; (iii) removal of the 5'-abasic terminal fragment, and (iv) repair synthesis to fill the gap by DNA polymerase beta; and (v) ligation to seal the remaining nick by DNA ligase 1 or a complex of DNA ligase 3 and X-ray repair cross-complementing 1. However, BER can involve the participation of other proteins as well, such as alternative DNA polymerases or one of several nonessential "auxiliary" factors. In addition, BER operates most efficiently when specific protein-protein coordination occurs. Furthermore, several BER protein activities have been shown to be regulated by posttranslational modification, and some of the physical protein interactions link BER to other DNA transaction pathways. In this review, we summarize the current state of the emerging complexities of mammalian BER, focusing on the growing number of reported protein-protein interactions and posttranslational modifications.
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PMID:Protein-protein interactions and posttranslational modifications in mammalian base excision repair. 1580 10

Repair of damaged DNA is of great importance in maintaining genome integrity, and there are several pathways for repair of damaged DNA in almost all organisms. Base excision repair (BER) is a main process for repairing DNA carrying slightly damaged bases. Several proteins are required for BER; these include DNA glycosylases, AP endonuclease, DNA polymerase, and DNA ligase. In some bacteria the single-stranded specific exonuclease, RecJ, is also involved in BER. In this research, six Chlamydiophila pneumoniae (C. pneumoniae) genes, encoding uracil DNA glycosylase (CpUDG), endonuclease IV (CpEndoIV), DNA polymerase I (CpDNApolI), endonuclease III (CpEndoIII), single-stranded specific exonuclease RecJ (CpRecJ), and DNA ligase (CpDNALig), were inserted into the expression vector pET28a. All proteins, except for CpDNALig, were successfully expressed in E. coli, and purified proteins were characterized in vitro. C. pneumoniae BER was reconstituted in vitro with CpUDG, CpEndoIV, CpDNApolI and E. coli DNA ligase (EcDNALig). After uracil removal by CpUDG, the AP site could be repaired by two BER pathways that involved in the replacement of either one (short patch BER) or multiple nucleotides (long patch BER) at the lesion site. CpEndoIII promoted short patch BER via its 5'-deoxyribophosphodiesterase (5'-dRPase) activity, while CpRecJ had little effect on short patch BER. The flap structure generated during DNA extension could be removed by the 5'-exonuclease activity of CpDNApolI. Based on these observations, we propose a probable mechanism for BER in C. pneumoniae.
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PMID:The mechanism of base excision repair in Chlamydiophila pneumoniae. 1608 68

Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T >> C > A > G, the same at the GUC site was T >> A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% G-->T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated approximately 60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo)-->T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with deltadeltaG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G(8-oxo)T 12-mer duplex with a deltadeltaG of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG(8-oxo)(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG(8-oxo)(AP*) sequence relative to (AP*)G(8-oxo)T. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG(8-oxo)(AP*) sequence context.
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PMID:Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells: tandem mutations and enhancement of G-->T transversions. 1609 91

The vaccinia virus E9 protein, the catalytic subunit of the DNA polymerase holoenzyme, is inherently distributive under physiological conditions, although infected cells contain a highly processive form of the enzyme. The viral A20 protein was previously characterized as a stoichiometric component of the processivity factor, and an interaction between A20 and E9 was documented in vivo. A20 has been shown to interact with D4, the virally encoded uracil DNA glycosylase (UDG), by yeast-two hybrid and in vitro analysis. Here we confirm that UDG and A20 interact in vivo and show that temperature-sensitive viruses with lesions in the D4R gene show a profound defect in DNA synthesis at the non-permissive temperature. Moreover, cytoplasmic extracts prepared from these infections lack processive polymerase activity in vitro, implicating D4 in the assembly or activity of the processive polymerase. Upon overexpression of 3xFLAG-UDG, A20, and E9 in various combinations, we purified dimeric and trimeric UDG-A20 and UDG-A20-polymerase complexes, respectively. These complexes are stable in 750 mm NaCl and can be further purified by Mono Q chromatography. Notably, the trimeric complex displays robust processive polymerase activity, and the dimeric complex can confer processivity on purified E9. Consistent with previous reports that the catalytic activity of UDG is dispensable for virus replication in tissue culture, we find that the role of UDG role in the polymerase complex is not diminished by mutations targeting residues involved in uracil recognition or excision. Our cumulative data support the conclusion that A20 and UDG form a heterodimeric processivity factor that associates with E9 to comprise the processive polymerase holoenzyme.
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PMID:Vaccinia virus uracil DNA glycosylase interacts with the A20 protein to form a heterodimeric processivity factor for the viral DNA polymerase. 1632 1

Chromatin in eukaryotic cells is folded into higher order structures of folded nucleosome filaments, and DNA damage occurs at all levels of this structural hierarchy. However, little is known about the impact of higher order folding on DNA repair enzymes. We examined the catalytic activities of purified human base excision repair (BER) enzymes on uracil-containing oligonucleosome arrays, which are folded primarily into 30 nm structures when incubated in repair reaction buffers. The catalytic activities of uracil DNA glycosylase (UDG) and apyrimidinic/apurinic endonuclease (APE) digest G:U mismatches to completion in the folded oligonucleosomes without requiring significant disruption. In contrast, DNA polymerase beta (Pol beta) synthesis is inhibited in a major fraction ( approximately 80%) of the oligonucleosome array, suggesting that single strand nicks in linker DNA are far more accessible to Pol beta in highly folded oligonucleosomes. Importantly, this barrier in folded oligonucleosomes is removed by purified chromatin remodeling complexes. Both ISW1 and ISW2 from yeast significantly enhance Pol beta accessibility to the refractory nicked sites in oligonucleosomes. These results indicate that the initial steps of BER (UDG and APE) act efficiently on highly folded oligonucleosome arrays, and chromatin remodeling may be required for the latter steps of BER in intact chromatin.
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PMID:Different structural states in oligonucleosomes are required for early versus late steps of base excision repair. 1757 92


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