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)

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by prematurely aged appearance. Genetic linkage analysis has placed the relevant gene in subchromosomal band 8p12. DNA polymerase beta gene has been mapped to chromosome 8p12-11 and thought to be involved in DNA repair and possibly in recombination. Somatic cells from WS patients exhibit chromosomal instability, a markedly reduced replicative life span and slow growth. The functions of DNA polymerase beta gene and its position prompted us to examine this gene in WS patients. We have found the novel DNA polymerase beta cDNA species in blood samples from WS patients, which contain 107 bp insertions or 87 bp deletions in the catalytic domain of DNA polymerase beta. These mutations change the structure of DNA polymerase beta and thus the capacity of the DNA repair system would be impaired, which may account for the high mutation rate observed in WS.
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PMID:Identification of mutations in DNA polymerase beta mRNAs from patients with Werner syndrome. 754 22

Werner syndrome (WS) is a rare autosomal recessive disorder of humans characterized by the premature onset and accelerated rate of development of several major age-related disorders. An aberration in DNA replication or repair is suggested by the evidence of genome instability. Since the structural gene for DNA polymerase beta maps within the region of the WS mutation on the short arm of chromosome 8 and is involved in both DNA repair and DNA replication, we evaluated its candidacy as the WS gene. Several independent lines of evidence did not support that hypothesis: (1) activity gels showed normal enzyme activity and electrophoretic mobility; (2) nucleotide sequence analysis of the entire coding region failed to reveal mutations (although indicated mistakes in the published sequence); (3) single-strand conformation polymorphism (SSCP) and heteroduplex analyses failed to reveal evidence of mutations in the promoter region; (4) a newly discerned polymorphism failed to reveal evidence of homozygosity by descent in a consanguineous patient; and 5) fluorescence in situ hybridization (FISH) analysis placed the DNA polymerase beta gene centromeric to D8S135 at 8p11.2 and thus beyond the region of peak LOD scores for WS.
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PMID:Evidence against DNA polymerase beta as a candidate gene for Werner syndrome. 816 25

Mammalian DNA polymerase beta is a crucial enzyme in cell genomic maintenance. Its structure is highly conserved. Some splice variants of beta-pol mRNA were observed. One alternative splice DNA polymerase beta mRNA, generated by 87 nt deletion (exon 11) in the catalytic domain of this enzyme, was suggested to be responsible for genomic instability in tumorigenesis and in genetic disorder (Werner syndrome). Here, we show that exon-11-deleted beta-pol mRNA is present in all examined normal and tumor tissues as well as in resting or PHA-stimulated peripheral-blood mononuclear cells. This finding proves that the presence of the exon-11 alternative splicing variant of beta-pol mRNA is not tumor-specific.
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PMID:Alternative splicing of DNA polymerase beta mRNA is not tumor-specific. 890 Apr 28

Positional cloning has already produced the sequences of more than 70 human genes associated with specific diseases. In addition to their medical importance, these genes are of interest as a set of human genes isolated solely on the basis of the phenotypic effect of the respective mutations. We analyzed the protein sequences encoded by the positionally cloned disease genes using an iterative strategy combining several sensitive computer methods. Comparisons to complete sequence databases and to separate databases of nematode, yeast, and bacterial proteins showed that for most of the disease gene products, statistically significant sequence similarities are detectable in each of the model organisms. Only the nematode genome encodes apparent orthologs with conserved domain architecture for the majority of the disease genes. In yeast and bacterial homologs, domain organization is typically not conserved, and sequence similarity is limited to individual domains. Generally, human genes complement mutations only in orthologous yeast genes. Most of the positionally cloned genes encode large proteins with several globular and nonglobular domains, the functions of some or all of which are not known. We detected conserved domains and motifs not described previously in a number of proteins encoded by disease genes and predicted functions for some of them. These predictions include an ATP-binding domain in the product of hereditary nonpolyposis colon cancer gene (a MutL homolog), which is conserved in the HS90 family of chaperone proteins, type II DNA topoisomerases, and histidine kinases, and a nuclease domain homologous to bacterial RNase D and the 3'-5' exonuclease domain of DNA polymerase I in the Werner syndrome gene product.
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PMID:Positionally cloned human disease genes: patterns of evolutionary conservation and functional motifs. 915

Individuals with mutations in the WRN gene suffer from Werner syndrome, a disease with early onset of many characteristics of normal aging. The WRN protein (WRNp) functions in DNA metabolism, as the purified polypeptide has both 3'-->5' helicase and 3'-->5' exonuclease activities. In this study, we have further characterized WRNp exonuclease activity by examining its ability to degrade double-stranded DNA substrates containing abnormal and damaged nucleo-tides. In addition, we directly compared the 3'-->5' WRNp exonuclease activity with that of exo-nuclease III and the Klenow fragment of DNA polymerase I. Our results indicate that the presence of certain abnormal bases (such as uracil and hypoxanthine) does not inhibit the exonuclease activity of WRNp, exo-nuclease III or Klenow, whereas other DNA modifications, including apurinic sites, 8-oxoguanine, 8-oxoadenine and cholesterol adducts, inhibit or block WRNp. The ability of damaged nucleo-tides to inhibit exonucleolytic digestion differs significantly between WRNp, exonuclease III and Klenow, indicating that each exonuclease has a distinct mechanism of action. In addition, normal and modified DNA substrates are degraded similarly by full-length WRNp and an N-terminal fragment of WRNp, indicating that the specificity for this activity lies mostly within this region. The biochemical and physiological significance of these results is discussed.
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PMID:Selective blockage of the 3'-->5' exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA. 1090 33

Werner syndrome is a Mendelian disorder of man that produces a number of manifestations resembling human aging. This disorder is caused by inactivation of the wrn gene, a member of the RecQ family of DNA helicases. The helicase and exonuclease activities of the Werner protein (WRN) suggest that it functions in DNA transactions, but the physiological function of WRN remains elusive. We present several lines of evidence that WRN interacts specifically with the p50 subunit of polymerase delta, the major DNA polymerase required for chromosomal DNA replication. P50, identified by yeast two-hybrid screening, interacts physically with the C terminus of WRN. Native WRN protein coimmunoprecipitates with p50 in a cellular fraction enriched in nucleolar proteins, and this immunocomplex also includes p125, the catalytic subunit of polymerase delta. In subcellular localization studies of cells transfected with WRN, p50 and p125 redistribute to the nucleolus and colocalize with WRN. These results suggest that one of the functions of WRN protein is to directly modify DNA replication via its interaction with p50 and abet dynamic relocalization of the DNA polymerase delta complexes within the nucleus.
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PMID:Werner protein recruits DNA polymerase delta to the nucleolus. 1102 36

Werner syndrome is a hereditary premature aging disorder characterized by genomic instability. Genetic analysis and protein interaction studies indicate that the defective gene product (WRN) may play an important role in DNA replication, recombination, and repair. DNA polymerase beta (pol beta) is a central participant in both short and long-patch base excision repair (BER) pathways, which function to process most spontaneous, alkylated, and oxidative DNA damage. We report here a physical interaction between WRN and pol beta, and using purified proteins reconstitute of a portion of the long-patch BER pathway to examine a potential role for WRN in this repair response. We demonstrate that WRN stimulates pol beta strand displacement DNA synthesis and that this stimulation is dependent on the helicase activity of WRN. In addition, a truncated WRN protein, containing primarily the helicase domain, retains helicase activity and is sufficient to mediate the stimulation of pol beta. The WRN helicase also unwinds a BER substrate, providing evidence that WRN plays a role in unwinding DNA repair intermediates. Based on these findings, we propose a novel mechanism by which WRN may mediate pol beta-directed long-patch BER.
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PMID:The Werner syndrome protein stimulates DNA polymerase beta strand displacement synthesis via its helicase activity. 1266 21

Werner syndrome patients are deficient in the Werner protein (WRN), which is a multifunctional nuclear protein possessing 3'-5' exonuclease and ATP-dependent helicase activities. Studies of Werner syndrome cells and biochemical studies of WRN suggest that WRN plays a role in several DNA metabolic pathways. WRN interacts with DNA polymerase beta (pol beta) and stimulates pol beta strand displacement synthesis on a base excision repair (BER) intermediate in a helicase-dependent manner. In this report, we examined the effect of the major human apurinic/apyrimidinic endonuclease (APE1) and of pol beta on WRN helicase activity. The results show that WRN alone is able to unwind several single strand break BER intermediates. However, APE1 inhibits WRN helicase activity on these intermediates. This inhibition is likely due to the binding of APE1 to nicked apurinic/apyrimidinic sites, suggesting that APE1 prevents the promiscuous unwinding of BER intermediates. This inhibitory effect was relieved by the presence of pol beta. A model involving the pol beta-mediated hand-off of WRN protein is proposed based on these results.
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PMID:Regulation of WRN helicase activity in human base excision repair. 1538 37

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

Genome instability is a characteristic of cancer and aging, and is a hallmark of the premature aging disorder Werner syndrome (WS). Evidence suggests that the Werner syndrome protein (WRN) contributes to the maintenance of genome integrity through its involvement in DNA repair. In particular, biochemical evidence indicates a role for WRN in base excision repair (BER). We have previously reported that WRN helicase activity stimulates DNA polymerase beta (pol beta) strand displacement synthesis in vitro. In this report we demonstrate that WRN exonuclease activity can act cooperatively with pol beta, a polymerase lacking 3'-5' proofreading activity. Furthermore, using small interference RNA technology, we demonstrate that WRN knockdown cells are hypersensitive to the alkylating agent methyl methanesulfonate, which creates DNA damage that is primarily repaired by the BER pathway. In addition, repair assays using whole cell extracts from WRN knockdown cells indicate a defect in long patch (LP) BER. These findings demonstrate that WRN plays a direct role in the repair of methylation-induced DNA damage, and suggest a role for both WRN helicase and exonuclease activities together with pol beta during LP BER.
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PMID:The Werner syndrome protein operates in base excision repair and cooperates with DNA polymerase beta. 1644 7


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