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)

Individuals with Xeroderma pigmentosum (XP) syndrome have a genetic predisposition to sunlight-induced skin cancer. Genetically different forms of XP have been identified by cell fusion. Cells of individuals expressing the classical form of XP (complementation groups A through G) are deficient in the nucleotide excision repair (NER) pathway. In contrast, the cells belonging to the variant class of XP (XPV) are NER-proficient and are only slightly more sensitive than normal cells to the killing action of UV light radiation. The XPV fibroblasts replicate damaged DNA generating abnormally short fragments either in vivo [A.R. Lehmann, The relationship between pyramidine dimers and replicating DNA in UV-irradiated human fibroblasts, Nucleic Acids Res. 7 (1979) 1901-1912; S.D. Park, J.E. Cleaver, Postreplication repair: question of its definition and possible alteration in Xeroderma pigmentosum cell strains, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 3927-3931.] or in vitro [S.M. Cordeiro, L.S. Zaritskaya, L.K. Price, W.K. Kaufmann, Replication fork bypass of a pyramidine dimer blocking leading strand DNA synthesis, J. Biol. Chem. 272 (1997) 13945-13954; D.L. Svoboda, L.P. Briley, J.M. Vos, Defective bypass replication of a leading strand cyclobutane thymine dimer in Xeroderma pigmentosum variant cell extracts, Cancer Res. 58 (1998) 2445-2448; I. Ensch-Simon, P.M. Burgers, J.S. Taylor, Bypass of a site-specific cis-syn thymine dimer in an SV40 vector during in vitro replication by HeLa and XPV cell-free extracts, Biochemistry 37 (1998) 8218-8226.], suggesting that in XPV cells, replication has an increased probability of being blocked at a lesion. Furthermore, extracts from XPV cells were found to be defective in translesion synthesis [A. Cordonnier, A.R. Lehmann, R.P.P. Fuchs, Impaired translesion synthesis in Xeroderma pigmentosum variant extracts, Mol. Cell. Biol. 19 (1999) 2206-2211.]. Recently, Masutani et al. [C. Masutani, M. Araki, A. Yamada, R. Kusomoto, T. Nogimori, T. Maekawa, S. Iwai, F. Hanaoka, Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity, EMBO J. 18 (1999) 3491-3501.] have shown that the XPV defect can be corrected by a novel human DNA polymerase, homologue to the yeast DNA polymerase eta, which is able to replicate past cyclobutane pyrimidine dimers in DNA templates. This review focuses on our current understanding of translesion synthesis in mammalian cells whose defect, unexpectedly, is responsible for the hypermutability of XPV cells and for the XPV pathology.
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PMID:Replication of damaged DNA: molecular defect in xeroderma pigmentosum variant cells. 1055 91

Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol(eta). Of the eukaryotic DNA polymerases, only human Pol(eta) and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol(eta) on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol(eta) misincorporates nucleotides with a frequency of approximately 10(-2)-10(-3), and importantly, hPol(eta) synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol(eta) may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.
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PMID:Fidelity of human DNA polymerase eta. 1071 43

Defects in the human gene XPV result in the variant form of the genetic disease xeroderma pigmentosum (XP-V). XPV encodes DNA polymerase eta, a novel DNA polymerase that belongs to the UmuC/DinB/Rad30 superfamily. This polymerase catalyzes the efficient and accurate translesion synthesis of DNA past cis-syn cyclobutane di-thymine lesions. In this report we present the cDNA sequence and expression profiles of the mouse XPV gene and demonstrate its ability to complement defective DNA synthesis in XP-V cells. The mouse XPV protein shares 80.3% amino acid identity and 86.9% similarity with the human XPV protein. The recombinant mouse XPV protein corrected the inability of XP-V cell extracts to carry out DNA replication, by bypassing thymine dimers on template DNA. Transfection of the mouse or human XPV cDNA into human XP-V cells corrected UV sensitivity. Northern blot analysis revealed that the mouse XPV gene is expressed ubiquitously, but at a higher level in testis, liver, skin and thymus compared to other tissues. Although the mouse XPV gene was not induced by UV irradiation, its expression was elevated approximately 4-fold during cell proliferation. These results suggest that DNA polymerase eta plays a role in DNA replication, though the enzyme is not essential for viability.
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PMID:Complementation of defective translesion synthesis and UV light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase eta. 1087 96

The Saccharomyces cerevisiae RAD30 gene encodes DNA polymerase eta. Humans possess two Rad30 homologs. One (RAD30A/POLH) has previously been characterized and shown to be defective in humans with the Xeroderma pigmentosum variant phenotype. Here, we report experiments demonstrating that the second human homolog (RAD30B), also encodes a novel DNA polymerase that we designate poliota. poliota, is a distributive enzyme that is highly error-prone when replicating undamaged DNA. At template G or C, the average error frequency was approximately 1 x 10(-2). Our studies revealed, however, a striking asymmetry in misincorporation frequency at template A and T. For example, template A was replicated with the greatest accuracy, with misincorporation of G, A, or C occurring with a frequency of approximately 1 x 10(-4) to 2 x 10(-4). In dramatic contrast, most errors occurred at template T, where the misincorporation of G was, in fact, favored approximately 3:1 over the correct nucleotide, A, and misincorporation of T occurred at a frequency of approximately 6.7 x 10(-1). These findings demonstrate that poliota is one of the most error-prone eukaryotic polymerases reported to date and exhibits an unusual misincorporation spectrum in vitro.
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PMID:poliota, a remarkably error-prone human DNA polymerase. 1088 58

Oxidative damage to DNA has been proposed to have a role in cancer and ageing. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and 7, 8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. In Saccharomyces cerevisiae, deletion of OGG1, encoding a DNA glycosylase that functions in the removal of 8-oxoG when paired with C, causes an increase in the rate of G:C to T:A transversions. The ogg1Delta msh2Delta double mutant displays a higher rate of CAN1S to can1r forward mutations than the ogg1Delta or msh2Delta single mutants, and this enhanced mutagenesis is primarily due to G:C to T:A transversions. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Poleta, that efficiently replicates DNA containing a cis-syn thymine-thymine (T-T) dimer by inserting two adenines across from the dimer. In humans, mutations in the yeast RAD30 counterpart, POLH, cause the variant form of xeroderma pigmentosum (XP-V), and XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. Here we show that yeast and human POLeta replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POLeta in the yeast ogg1Delta mutant. Our results suggest an additional role for Poleta in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.
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PMID:Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase eta. 1093 95

Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribose) polymerases (PARPs). PARP-1, the best understood and until recently the only known member of this family, is a DNA damage signal protein catalyzing its automodification with multiple, variably sized ADP-ribose polymers that may contain up to 200 residues and several branching points. Through these polymers, PARP-1 can interact noncovalently with other proteins and alter their functions. Here we report the discovery of a poly(ADP-ribose)-binding sequence motif in several important DNA damage checkpoint proteins. The 20-amino acid motif contains two conserved regions: (i) a cluster rich in basic amino acids and (ii) a pattern of hydrophobic amino acids interspersed with basic residues. Using a combination of alanine scanning, polymer blot analysis, and photoaffinity labeling, we have identified poly(ADP-ribose)-binding sites in the following proteins: p53, p21(CIP1/WAF1), xeroderma pigmentosum group A complementing protein, MSH6, DNA ligase III, XRCC1, DNA polymerase epsilon, DNA-PK(CS), Ku70, NF-kappaB, inducible nitric-oxide synthase, caspase-activated DNase, and telomerase. The poly(ADP-ribose)-binding motif was found to overlap with five important functional domains responsible for (i) protein-protein interactions, (ii) DNA binding, (iii) nuclear localization, (iv) nuclear export, and (v) protein degradation. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions.
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PMID:Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins. 1101 34

The genetic disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD) are all associated with defects in nucleotide excision repair (NER) of DNA damage. Their clinical features are very different, however, XP being a highly cancer-prone skin disorder, whereas CS and TTD are cancer-free multisystem disorders. All three are genetically complex, with at least eight complementation groups for XP (XP-A to -G and variant), five for CS (CS-A, CS-B, XP-B, XP-D, and XP-G), and three for TTD (XP-B, XP-D, and TTD-A). With the exception of the variant, the products of the XP genes are proteins involved in the different steps of NER, and comprise three damage-recognition proteins, two helicases, and two nucleases. The two helicases, XPB and XPD, are components of the basal transcription factor TFIIH, which has a dual role in NER and initiation of transcription. Different mutations in these genes can affect NER and transcription differentially, and this accounts for the different clinical phenotypes. Mutations resulting in defective repair without affecting transcription result in XP, whereas if transcription is also affected, TTD is the outcome. CS proteins are only involved in transcription-coupled repair, a subpathway of NER in which damage in the transcribed strands of active genes is rapidly and preferentially repaired. Current evidence suggests that they also have an important but not essential role in transcription. The variant form of XP is defective in a novel DNA polymerase, which is able to synthesise DNA past UV-damaged sites.
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PMID:Xeroderma pigmentosum and related disorders: defects in DNA repair and transcription. 1103 99

Xeroderma pigmentosum variant (XP-V) cells are defective in bypass replication of UVC-induced thymine dimers in DNA because they lack a novel DNA polymerase (polymerase eta). In this study the effects of UVC on S phase cells were compared in fibroblasts derived from normal donors (IDH4) and XP-V patients (CTag) and immortalized by expression of the SV40 large T antigen. These transformed fibroblasts did not activate the G(1) checkpoint or inhibit replicon initiation when damaged by UVC or gamma-rays. The transformed XP-V cells (CTag) retained the increased sensitivity to UVC-induced inhibition of DNA strand growth previously observed with their diploid counterpart. Cell cycle progression analyses showed that CTag cells displayed a stronger S phase delay than transformed fibroblasts from normal individuals (IDH4) after treatment with only 2 J/m(2) UVC. Low doses of UVC also caused a lag in CTag cell proliferation. The extent of replication of an episomal DNA (pSV011), not previously exposed to radiation, was measured after the host cells were irradiated with 1-3 J/m(2) UVC. Replication of pSV011 was barely affected in irradiated IDH4 cells. Plasmid replication was inhibited by 50% in irradiated CTag cells and this inhibition could not be accounted for by increased killing of host cells by UVC. These results suggest that even in transformed cells UVC induces DNA damage responses that are reflected in transient cell cycle arrest, delay in proliferation and inhibition of episomal DNA replication. These responses are enhanced in CTag cells, presumably because of their bypass replication defect. The accumulation of replication complexes blocked at thymine dimers and extended single-stranded regions in chromosomal DNA might sequester replication factors that are needed for plasmid and chromosomal replication. Alternatively, aberrant replication structures might activate a signal transduction pathway that down-regulates DNA synthesis.
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PMID:Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant. 1118 43

Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerase delta and epsilon, is involved in both DNA replication and repair. Previous studies in vitro have demonstrated the requirement of PCNA in the resynthesis step of nucleotide excision repair (NER) and base excision repair (BER). Using a native chromatin template isolated under near physiological conditions, we have analysed the involvement of PCNA in the BER pathway in different NER defective human cell lines. The repair sites and PCNA were visualized by indirect immunolabelling followed by fluorescence microscopy. The results indicate that exposure to X-rays triggers the induction of PCNA in all the three human fibroblast cell lines studied, namely normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). In all the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient XP-A cells suggests that the X-ray-induced lesions are largely repaired via the BER pathway involving PCNA as one of the key components of this pathway. X-ray-induced repair synthesis was greatly inhibited by treatment of cells with DNA polymerase inhibitors aphidicolin and cytosine arabinoside. Interestingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be required for the BER pathway at a step preceding the resynthesis step.
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PMID:Analysis of repair and PCNA complex formation induced by ionizing radiation in human fibroblast cell lines. 1132 Jan 48

DNA polymerase eta (Poleta) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Poleta in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV). Both Saccharomyces cerevisiae and human Poleta efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer. Interestingly, in the fission yeast Schizosaccharomyces pombe, the eso1(+) encoded protein is comprised of two domains, wherein the NH(2) terminus is highly homologous to Poleta, and the COOH terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase. Here we characterize the DNA polymerase activity of S. pombe GST-Eso1 fusion protein and a truncated version containing only the Poleta domain. Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of approximately 10(-2) to 10(-3). We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well. Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Poleta. The possible role of Ctf7 fusion with Poleta in the replication of Cohesin-bound DNA sequences is discussed.
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PMID:Fidelity and damage bypass ability of Schizosaccharomyces pombe Eso1 protein, comprised of DNA polymerase eta and sister chromatid cohesion protein Ctf7. 1155 52


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