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)

The sliding clamp, PCNA, of the archaeon Sulfolobus solfataricus P2 is a heterotrimer of three distinct subunits (PCNA1, 2, and 3) that assembles in a defined manner. The PCNA heterotrimer, but not individual subunits, stimulates the activities of the DNA polymerase, DNA ligase I, and the flap endonuclease (FEN1) of S. solfataricus. Distinct PCNA subunits contact DNA polymerase, DNA ligase, or FEN1, imposing a defined architecture at the lagging strand fork and suggesting the existence of a preformed scanning complex at the fork. This provides a mechanism to tightly couple DNA synthesis and Okazaki fragment maturation. Additionally, unique subunit-specific interactions between components of the clamp loader, RFC, suggest a model for clamp loading of PCNA.
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PMID:A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solfataricus. 1253 40

Single-strand DNA interruptions (SSIs) are produced during the process of base excision repair (BER). Through biochemical studies, two SSI repair subpathways have been identified: a pathway mediated by DNA polymerase beta (Pol beta) and DNA ligase III (Lig III), and a pathway mediated by DNA polymerase delta/epsilon (Pol delta/epsilon) and DNA ligase I (Lig I). In addition, the existence of another pathway, mediated by Pol beta and DNA Lig I, has been suggested. Although each pathway may play a unique role in cellular DNA damage response, the functional implications of SSI repair by these three pathways are not clearly understood. To obtain a better understanding of the functional relevance of SSI repair by these pathways, we investigated the involvement of each pathway by monitoring the utilization of DNA ligases in cell-free extracts. Our results suggest that the majority of SSIs produced during the repair of alkylated DNA bases are repaired by the pathway mediated by Pol beta and either Lig I or Lig III, although some SSIs are repaired by Pol delta/epsilon and Lig I. At a cellular level, we found that Lig III over-expression increased the resistance of cells to DNA-damaging agents, while Lig I over-expression had little effect. Thus, repair pathways mediated by Lig III may have a role in the regulation of cellular sensitivity to DNA-damaging agents.
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PMID:Repair of single-strand DNA interruptions by redundant pathways and its implication in cellular sensitivity to DNA-damaging agents. 1462 36

Radical oxygen species (ROS) generate various modified DNA bases. Among them 8-oxo-7,8-dihydroguanine (8oxoG) is the most abundant and seems to play a major role in mutagenesis and in carcinogenesis. 8oxoG is removed from DNA by the specific glycosylase OGG1. An additional post-replication repair is needed to correct the 8oxoG/A mismatches that are produced by persistent 8oxoG residues. This review is focused on the mechanisms of base excision repair (BER) of this oxidized base. It is shown that, in vitro, efficient and complete repair of 8oxoG/C pairs requires a core of four proteins, namely OGG1, APE1, DNA polymerase (Pol) beta, and DNA ligase I. Repair occurs predominantly by one nucleotide replacement reactions (short-patch BER) and Pol beta is the polymerase of election for the resynthesis step. However, alternative mechanisms can act on 8oxoG residues since Pol beta-null cells are able to repair these lesions. 8oxoG/A mismatches are repaired by human cell extracts via two BER events which occur sequentially on the two strands. The removal of the mismatched adenine is followed by preferential insertion of a cytosine leading to the formation of 8oxoG/C pairs which are then corrected by OGG1-mediated BER. Both repair events are inhibited by aphidicolin, suggesting that a replicative DNA polymerase is involved in the repair synthesis step. We propose that Pol delta/epsilon-mediated BER (long-patch BER) is the mode of repair when lesions persist or are formed at replication. Finally, we address the issues of the relative contribution of the two BER pathways to oxidative damage repair in vivo and the possible role of BER gene variants as cancer susceptibility genes.
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PMID:8-Oxoguanine DNA damage: at the crossroad of alternative repair pathways. 1463 50

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.
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PMID:Repair of abasic sites in DNA. 1463 52

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.
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PMID:Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae. 1528 91

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase ot-primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase ca and recruits DNA polymerase S and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase 8, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase E normally replicates this strand,but under conditions of dysfunction, DNA polymerase 8 may substitute.
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PMID:DNA polymerases that propagate the eukaryotic DNA replication fork. 1581 31

DNA single-strand breaks containing 3'-8-oxoguanine (3'-8-oxoG) ends can arise as a consequence of ionizing radiation and as a result of DNA polymerase infidelity by misincorporation of 8-oxodGMP. In this study we examined the mechanism of repair of 3'-8-oxoG within a single-strand break using purified base excision repair enzymes and human whole cell extracts. We find that 3'-8-oxoG inhibits ligation by DNA ligase IIIalpha or DNA ligase I, inhibits extension by DNA polymerase beta and that the lesion is resistant to excision by DNA glycosylases involved in the repair of oxidative lesions in human cells. However, we find that purified human AP-endonuclease 1 (APE1) is able to remove 3'-8-oxoG lesions. By fractionation of human whole cell extracts and immunoprecipitation of fractions containing 3'-8-oxoG excision activity, we further demonstrate that APE1 is the major activity involved in the repair of 3'-8-oxoG lesions in human cells and finally we reconstituted repair of the 3'-8-oxoG-containing oligonucleotide duplex with purified human enzymes including APE1, DNA polymerase beta and DNA ligase IIIalpha.
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PMID:APE1-dependent repair of DNA single-strand breaks containing 3'-end 8-oxoguanine. 1583 93

The human checkpoint sensor and alternative clamp Rad9-Rad1-Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9-Rad1-Hus1 with MutY DNA glycosylase, DNA polymerase beta and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9-Rad1-Hus1 complex, thus enhancing the quality control of DNA.
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PMID:The human checkpoint sensor and alternative DNA clamp Rad9-Rad1-Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery. 1587 98

Arsenic is an established human carcinogen. However, there has been much controversy about the shape of the arsenic response curve, particularly at low doses. This controversy has been exacerbated by the fact that the mechanism(s) of arsenic carcinogenesis are still unclear and because there are few satisfactory animal models for arsenic-induced carcinogenesis. Recent epidemiological studies have shown that the relative risk for cancer among populations exposed to <or=60 ppb As in their drinking water is often lower than the risk for the unexposed control population. We have found that treatment of human keratinocyte and fibroblast cells with 0.1 to 1 microM arsenite (As(III)) also produces a low dose protective effect against oxidative stress and DNA damage. This response includes increased transcription, protein levels and enzyme activity of several base excision repair genes, including DNA polymerase beta and DNA ligase I. At higher concentrations (> 10 microM), As induces down-regulation of DNA repair, oxidative DNA damage and apoptosis. This low dose adaptive (protective) response by a toxic agent is known as hormesis and is characteristic of many agents that induce oxidative stress. A mechanistic model for arsenic carcinogenesis based on these data would predict that the low dose risk for carcinogenesis should be sub-linear. The threshold dose where toxicity outweighs protection is hard to predict based on in vitro dose response data, but might be estimated if one could determine the form (metabolite) and concentration of arsenic responsible for changes in gene regulation in the target tissues.
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PMID:Arsenic, mode of action at biologically plausible low doses: what are the implications for low dose cancer risk? 1599

Accumulating evidence suggests that the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, known to be a sensor of DNA damage, is also a component of DNA repair systems. Recent results show that 9-1-1 interacts with several base excision repair proteins. It binds the DNA glycosylase MutY homolog, and stimulates DNA polymerase beta, flap endonuclease 1, and DNA ligase I. 9-1-1 resembles proliferating cell nuclear antigen (PCNA), which stimulates some of these same repair enzymes, and is loaded onto DNA in a similar manner. The complex of 9-1-1 with DNA ligase I can be immunoprecipitated from human cells. Moreover, UV irradiation stimulates 9-1-1.ligase I complex formation, suggesting a role for 9-1-1 in DNA repair. Examining the nature of 9-1-1 interaction with DNA ligase I, we show that there is a similar degree of stimulation on ligation substrates with different structures, and that there is specificity for DNA ligase I. 9-1-1 improves the binding of DNA ligase I to nicked double strand DNA. Furthermore, although high concentrations of casein kinase II strongly inhibits DNA ligase I activity, it does not affect the ability of 9-1-1 to stimulate. This suggests that 9-1-1 is also an activator of DNA ligase I during DNA damage. Unlike PCNA, 9-1-1 stimulates DNA ligase I activity to the same extent on both linear and circular substrates, indicating that encirclement is not a requirement for stimulation. These data are consistent with a direct role for 9-1-1 in DNA repair, but possibly employing a different mechanism than PCNA.
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PMID:Mechanism of stimulation of human DNA ligase I by the Rad9-rad1-Hus1 checkpoint complex. 1673 26


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