Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The formation of DNA adducts in human HepG2 cells and human hepatocytes exposed to 14C-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined using Accelerator Mass Spectrometry (AMS). PhIP generated DNA adducts in a linear dose-dependent manner between 100 pM and 20 micro M. Co-treatment with the dietary isothiocyanate, sulforaphane (SFN, 1-10 micro M), or the flavonoid, quercetin (5-20 micro M), significantly reduced the level of PhIP-DNA adducts in a dose-dependent manner. The degree of protection was dependent on PhIP concentration, i.e. after 100 pM PhIP exposure, SFN or quercetin reduced adduct levels to below the limit of detection (0.15 amol PhIP/ micro g DNA) but at higher PhIP exposure (10 nM and 1 micro M), the protection was 60 and 10%, respectively. The involvement of phase I, phase II and DNA repair enzymes in this protection against PhIP-DNA adduct formation was investigated using real-time RT-PCR and enzyme activity assays. In intact HepG2 cells, quercetin inhibited cytochrome P450 (CYP)1A2, the main phase I enzyme responsible for PhIP bioactivation. In contrast, SFN induced phase II detoxification enzymes, UDP-glucuronosyltransferase 1A1 and glutathione S-transferase A1 mRNA expression. SFN and quercetin showed no effect on DNA repair, neither in terms of the level of PhIP-DNA adducts, when cells were treated with phytochemicals after the carcinogen exposure, nor the regulation of mRNA expression of two DNA repair enzymes, apurinic endonuclease and DNA polymerase beta. This study indicates that dietary isothiocyanates and flavonoids modulate phase I and phase II enzyme expression, hence increasing the rate of detoxification of the dietary carcinogen PhIP in human HepG2 cells but do not affect the rate of PhIP-DNA adduct repair. The formation of PhIP-DNA adducts in human hepatocytes was also dose-dependent with PhIP-concentration and the levels of protection by SFN or quercetin were up to 60% after 10 nM PhIP treatment, but showed large inter-individual variation with no observed protection in some individuals.
 ...
PMID:Sulforaphane and quercetin modulate PhIP-DNA adduct formation in human HepG2 cells and hepatocytes. 1294 46

DNA base excision repair (BER) constitutes a major mechanism to restore the integrity of the genome following modifications of nucleobases. Although it is well established that poly(ADP-ribosylation) facilitates BER, the mechanism of this stimulation has remained unknown. Previous observations suggested that poly(ADP-ribose), which is synthesised from NAD(+), could serve as a unique source of ATP required for the ligation step in BER. This pathway of ATP generation is thought to compensate ATP shortage and relies on the release of pyrophosphate during DNA repair synthesis. Here, we present evidence that, in situations of cellular energy depletion, the synthesis of poly(ADP-ribose) is indeed stimulated. Simultaneously, single nucleotide repair is reduced. Rather, the number of nucleotides incorporated by DNA polymerase beta (Pol beta) during DNA repair synthesis is increased. Using a reconstituted system including the recombinant BER proteins Pol beta, AP endonuclease 1 (APE 1), X-ray repair cross-complementing group-1 (XRCC1), DNA ligase III (Lig III), flap endonuclease 1 (FEN 1), and poly(ADP-ribose) polymerase-1 (PARP-1), it is demonstrated that in the absence of ATP, both long patch DNA synthesis by Pol beta and poly(ADP-ribosylation) catalysed by PARP-1 are stimulated. Consequently, the preferred use of either long patch or single nucleotide BER depends on the availability of ATP. It is proposed that long patch BER is required for ATP generation from poly(ADP-ribose) and, therefore, predominant under conditions of ATP shortage.
 ...
PMID:ATP-dependent selection between single nucleotide and long patch base excision repair. 1367 48

The Ape1 protein initiates the repair of apurinic/apyrimidinic sites during mammalian base excision repair (BER) of DNA. Ape1 catalyzes hydrolysis of the 5'-phosphodiester bond of abasic DNA to create nicks flanked by 3'-hydroxyl and 5'-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) beta catalyzes both DNA synthesis at the 3'-hydroxyl terminus and excision of the 5'-dRP moiety prior to completion of BER by DNA ligase. During BER, Ape1 recruits pol beta to the incised apurinic/apyrimidinic site and stimulates 5'-dRP excision by pol beta. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Ape1 to modulate the deoxynucleotidyltransferase and 5'-dRP lyase activities of pol beta. We report here that Ape1 stimulates 5'-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Ape1, in which conditions that support DNA synthesis by pol beta also enhance 5'-dRP excision. Ape1 modulates the gap-filling activity of pol beta by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Ape1 protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol beta. However, this mutant protein retained the ability to stimulate 5'-dRP excision by pol beta. Simultaneous monitoring of 5'-dRP excision and DNA synthesis by pol beta demonstrated that the 5'-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER.
 ...
PMID:Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinic endonuclease 1. 1507 79

Human apurinic/apyrimidinic endonuclease (APE1) is an enzyme of DNA base excision repair (BER) which catalyzes endonucleolytic cleavage immediately 5' to abasic (AP) sites. APE1 has long been thought to act on AP sites only in double stranded (ds) DNA, in order to generate the appropriate site for insertion of the correct nucleotide of DNA repair synthesis effected by DNA polymerase beta. We now present evidence that APE1 also acts on AP sites in single-stranded (ss) DNA. The catalytic efficiency of this activity (defined within as k(cat)/Km) is approximately 20-fold less than the activity against AP sites in ds DNA, with the disparity stemming largely from a difference in Km. Similar to its action on AP sites in ds DNA, catalysis of endonucleolytic cleavage of ss DNA by APE1 is Mg(2+) dependent, DNA N-glycosylase independent, and requires an active site aspartate. In contrast to its activity against AP sites in ds DNA, APE1 does not display product inhibition when acting on an AP site in ss DNA. We suggest that this novel activity is related to the processing of DNA N-glycosylase initiated BER in ss DNA perhaps during replication and/or transcription.
 ...
PMID:Human AP endonuclease (APE1) demonstrates endonucleolytic activity against AP sites in single-stranded DNA. 1508 14

Base excision repair (BER) is a defense system that protects cells from deleterious effects secondary to modified or missing DNA bases. BER is known to involve apurinic/apyrimidinic endonuclease (APE) and DNA polymerase ss (ss-pol) among other enzymes, and recent studies have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) also plays a role by virtue of its binding to BER intermediates. The main role of APE is cleavage of the DNA backbone at abasic sites, and the enzyme also can catalyze 3'- to 5'-exonuclease activity at the cleaved abasic site. Photocross-linking studies with mouse embryonic fibroblast (MEF) cell extracts described here indicated that APE and PARP-1 interact with the same APE-cleaved abasic site BER intermediate. The model BER intermediate used includes a synthetic abasic site sugar, i.e. tetrahydrofuran (THF), in place of the natural deoxyribose. APE cross-linked efficiently with this intermediate, but not with a molecule lacking the 5'-THF phosphate group, and the same property was demonstrated for PARP-1. The addition of purified APE to the MEF extract reduced the amount of PARP-1 cross-linked to the BER intermediate, suggesting that APE can compete with PARP-1. APE and PARP-1 were antagonists of each other in in vitro BER related reactions on this model BER intermediate. These results suggest that PARP-1 and APE can interact with the same BER intermediate and that competition between these two proteins may influence their respective BER related functions.
 ...
PMID:AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate. 1513 26

Two unusual enzymatic activities are required for the realization of a single molecule sequencing: a polymerase for copying a deoxyribonuclease (DNA) target into complementary flurophore-labeled DNA, and an exonuclease for the successive hydrolysis of the completely dye-labeled DNA. Recently, we found that the wild-type Klenow fragment of Escherichia coli DNA polymerase I is well-suited for the synthesis of DNA in a reaction set-up that contains exclusively specific rhodamine-labeled analogs of the natural pyrimidine nucleotides (dCTP and dTTP). This protocol describes the procedure used for the preparation of DNA that is labeled at all pyrimidine bases of one strand, as well as an example of enzymatic downstream processing of the DNA product.
 ...
PMID:High-density labeling of DNA for single molecule sequencing. 1519 7

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.
 ...
PMID:Regulation of WRN helicase activity in human base excision repair. 1538 37

We examined interactions between base excision repair (BER) DNA intermediates and purified human BER enzymes, DNA polymerase beta (pol beta), apurinic/apyrimidinic endonuclease (APE1) and poly(ADP-ribose) polymerase-1 (PARP-1). Studies under steady-state conditions with purified BER enzymes and BER substrates have already demonstrated interplay between these BER enzymes that is sensitive to the respective concentrations of each enzyme. Therefore, in this study, using conditions of enzyme excess over substrate DNA, we further examine the question of interplay between BER enzymes on BER intermediates. The results reveal several important differences compared with data obtained using steady-state assays. Excess PARP-1 antagonizes the action of pol beta, producing a complete block of long patch BER strand-displacement DNA synthesis. Surprisingly, an excess of APE1 stimulates strand-displacement DNA synthesis by pol beta, but this effect is blocked by PARP-1. The APE1 exonuclease function appears to be modulated by the other BER proteins. Excess APE1 over pol beta may allow APE1 to perform both exonuclease function and stimulation of strand-displacement DNA synthesis by pol beta. This enables pol beta to mediate long patch sub-pathway. These results indicate that differences in the stoichiometry of BER enzymes may regulate BER.
 ...
PMID:Human base excision repair enzymes apurinic/apyrimidinic endonuclease1 (APE1), DNA polymerase beta and poly(ADP-ribose) polymerase 1: interplay between strand-displacement DNA synthesis and proofreading exonuclease activity. 1573 42

DNA polymerase lambda (Pol lambda) is a novel enzyme of the family X of DNA polymerases. Pol lambda has some properties in common with DNA polymerase beta (Pol beta). The substrate properties of Pol lambda were compared to Pol beta using DNAs mimicking short-patch (SP) and long-patch (LP) base excision repair (BER) intermediates as well as recessed template primers. In the present work, the influence of several BER proteins such as flap-endonuclease-1 (FEN1), PCNA, and apurinic/apyrimidinic endonuclease-1 (APE1) on the activity of Pol lambda was investigated. Pol lambda is unable to catalyze strand displacement synthesis using nicked DNA, although this enzyme efficiently incorporates a dNMP into a one-nucleotide gap. FEN1 and PCNA stimulate the strand displacement activity of Pol lambda. FEN1 processes nicked DNA, thus removing a barrier to Pol lambda DNA synthesis. It results in a one-nucleotide gapped DNA molecule that is a favorite substrate of Pol lambda. Photocrosslinking and functional assay show that Pol lambda is less efficient than Pol beta in binding to nicked DNA. APE1 has no influence on the strand displacement activity of Pol lambda though it stimulates strand displacement synthesis catalyzed with Pol beta. It is suggested that Pol lambda plays a role in the SP BER rather than contributes to the LP BER pathway.
 ...
PMID:Comparison of functional properties of mammalian DNA polymerase lambda and DNA polymerase beta in reactions of DNA synthesis related to DNA repair. 1597 54

As part of a systematic study of the effects of phytochemicals beyond antioxidation on cancer prevention, we investigated whether naringenin (NR), a citrus flavonoid, stimulates DNA repair following oxidative damage in LNCaP human prostate cancer cells. The 8-hydroxydeoxyguanosine (8-OH-dG) to deoxyguanosine (dG) ratio was measured after cells were treated with 200 micromol/L of ferrous sulfate in serum-free medium followed by NR exposure for 24 h in growth medium. The results demonstrated that exposure to 10-80 micromol/L of NR led to a significant decrease in the ratio of 8-OH-dG to 10(6) dG. Because cells were treated with NR after ferrous sulfate was removed, we conclude that we demonstrated an effect on DNA repair beyond antioxidation. In support of this conclusion, we determined the induction of mRNA expression over time after oxidative stress followed by NR administration of three major enzymes in the DNA base excision repair (BER) pathway: 8-oxoguanine-DNA glycosylase 1 (hOGG1), apurinic/apyrimidinic endonuclease and DNA polymerase beta (DNA poly beta). hOGG1 and DNA poly beta mRNA expression in cells after 24-h exposure to NR was increased significantly compared with control cells without NR. The intracellular concentration of NR after exposure to 80 micromol/L was 3 pmol/mg protein, which is physiologically achievable in tissues. In conclusion, the cancer-preventive effects of citrus fruits demonstrated in epidemiological studies may be due in part to stimulation of DNA repair by NR, which by stimulating BER processes may prevent mutagenic changes in prostate cancer cells.
 ...
PMID:The citrus flavonoid naringenin stimulates DNA repair in prostate cancer cells. 1611 81


<< Previous 1 2 3 4 5 6 7 8 9 10