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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG). MutY is a base excision repair (BER) glycosylase that removes misincorporated adenine residues from OG:A mispairs, as well as G:A and C:A mispairs. We have previously shown that, under conditions of low MutY concentrations relative to an OG:A or G:A substrate, the time course of the adenine glycosylase reaction exhibits biphasic kinetic behavior due to slow release of the DNA product by MutY. The dissociation of MutY from its product may require the recruitment of other proteins from the BER pathway, such as an apurinic-apyrimidinic (AP) endonuclease, as turnover-enhancing cofactors. The effect of the AP endonucleases endonuclease IV (Endo IV), exonuclease III (Exo III), and Ape1 on the reaction kinetics of MutY with G:A- and OG:A-containing substrates was investigated. The effect of the glycosylases UDG and MutM and the DNA polymerase pol I was also characterized. Endo IV and Exo III, unlike Ape1, UDG, and pol I, greatly enhance the rate of product release with a G:A substrate, whereas the rate constant for the adenine removal step remains unchanged. Furthermore, the turnover rate with a truncated form of MutY, Stop 225, which lacks 125 amino acids of the C terminus, is unaffected by the presence of Endo IV or Exo III. These results constitute the first evidence of an interaction between the MutY-product DNA complex and Endo IV or Exo III. Furthermore, they suggest a role for the C-terminal domain of MutY in mediating this interaction.
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PMID:Escherichia coli apurinic-apyrimidinic endonucleases enhance the turnover of the adenine glycosylase MutY with G:A substrates. 1196 Sep 95

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.
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PMID:Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins. 1200 Aug 32

There are numerous studies documenting the increase of oxidative DNA damage in the nuclei and mitochondria of senescing cells as well as in tissues of aging animals. Here, we show that in IMR 90 human diploid fibroblasts, DNA repair activity is robust in both nuclear and mitochondrial extracts, however, the levels of activity differed against the three substrates tested. In extracts, cleavage of the 8-oxoguanine substrate, and to a lesser extent the dihydrouracil-containing substrate, occurred in a concerted reaction between the DNA glycosylases and the second enzyme in the reaction, hAPE. Cleavage of both the furan and the dihydrouracil-containing substrates was unchanged when nuclear extracts from early and late passage cells were compared. However, cleavage of the 8-oxoguanine substrate was substantially reduced in the nuclear extracts from late passage cells and significantly reduced transcription from the hOGG1 gene was observed. When mitochondrial extracts were examined, activity on all three substrates was significantly reduced, with the reduction in hAPE activity being the most marked. The reduction in cleavage of the furan substrate was not simply due to inactive mitochondrial AP endonuclease but a substantially reduced amount of hAPE protein; transcription from the hAPE gene was also reduced. Confocal microscopic analysis confirmed that hAPE was present in the mitochondria of early passage cells but greatly reduced in the mitochondria of late passage cells. Cytoplasmic extracts from late passage fibroblasts also showed reduced activity with all three substrates suggesting that the residual hAPE, and activities that recognized dihydrouracil, were preferentially targeted to the nuclei. Taken together the data support the concept that the increase in oxidative damage in the mitochondrial DNA of senescing cells and tissues from aging animals is due to reduced base excision repair activity.
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PMID:Decline of nuclear and mitochondrial oxidative base excision repair activity in late passage human diploid fibroblasts. 1276 47

XRCC1 participates in DNA single strand break and base excision repair (BER) to preserve genetic stability in mammalian cells. XRCC1 participation in these pathways is mediated by its interactions with several of the acting enzymes. Here, we report that XRCC1 interacts physically and functionally with hOGG1, the human DNA glycosylase that initiates the repair by BER of the mutagenic oxidized base 8-oxoguanine. This interaction leads to a 2- to 3-fold stimulation of the DNA glycosylase activity of hOGG1. XRCC1 stimulates the formation of the hOGG1 Schiff-base DNA intermediate without interfering with the endonuclease activity of APE1, the second enzyme in the pathway. On the contrary, the stimulation in the appearance of the incision product seems to reflect the addition of the effects of XRCC1 on the two first enzymes of the pathway. The data presented support a model by which XRCC1 will pass on the DNA intermediate from hOGG1 to the endonuclease APE1. This results in an acceleration of the overall repair process of oxidized purines to yield an APE1-cleaved abasic site, which can be used as a substrate by DNA polymerase beta. More importantly, the results unveil a highly coordinated mechanism by which XRCC1, through its multiple protein-protein interactions, extends its orchestrating role from the base excision step to the resealing of the repaired DNA strand.
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PMID:Role of XRCC1 in the coordination and stimulation of oxidative DNA damage repair initiated by the DNA glycosylase hOGG1. 1293 15

Despite the progress in understanding the base excision repair (BER) pathway it is still unclear why known mutants deficient in DNA glycosylases that remove oxidised bases are not sensitive to oxidising agents. One of the back-up repair pathways for oxidative DNA damage is the nucleotide incision repair (NIR) pathway initiated by two homologous AP endonucleases: the Nfo protein from Escherichia coli and Apn1 protein from Saccharomyces cerevisiae. These endonucleases nick oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to repair of the remaining 5'-dangling nucleotide. NIR provides an advantage compared to DNA glycosylase-mediated BER, because AP sites, very toxic DNA glycosylase products, do not form. Here, for the first time, we have characterised the substrate specificity of the Apn1 protein towards 5,6-dihydropyrimidine, 5-hydroxy-2'-deoxyuridine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine deoxynucleotide. Detailed kinetic comparisons of Nfo, Apn1 and various DNA glycosylases using different DNA substrates were made. The apparent K(m) and kcat/K(m) values of the reactions suggest that in vitro DNA glycosylase/AP lyase is somewhat more efficient than the AP endonuclease. However, in vivo, using cell-free extracts from paraquat-induced E.coli and from S.cerevisiae, we show that NIR is one of the major pathways for repair of oxidative DNA base damage.
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PMID:Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases. 1457 22

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK(-) osteosarcoma cells that had been depleted of mtDNA (rho(0)) or not (wt). Despite the total absence of mtDNA in the rho(0) cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in rho(0) relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase gamma activities were more affected, 65 and 45% lower, respectively, in rho(0) mitochondria. Overall BER activity in lysates was also about 65% reduced in rho(0) mitochondria. To identify the limiting deficiencies in BER of rho(0) mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase gamma. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase gamma. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in rho(0) mitochondria. While nuclear BER protein levels and activities were generally not altered in rho(0) cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in rho(0) cells, and not a general dysfunction of rho(0) cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.
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PMID:DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA. 1510 86

Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 microg/ml) and HeLa cells were irradiated by (60)Co gamma-rays, and abasic (AP) sites and endonuclease (Endo)III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10(6) base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10(6) bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10(6) bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation.
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PMID:Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in gamma-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay. 1530 65

The tumor suppressor p53 plays an important role in the regulation of cellular response to DNA damage. Recent studies suggest that p53 is able to bind DNA with certain structural alterations in a sequence-independent manner and to interact with several molecules involved in DNA repair. This study was undertaken to test the hypothesis that p53 may participate in sensing oxidative DNA damage, the most frequently occurring spontaneous DNA lesion, and modulate its repair by the base excision repair (BER) machinery. Using synthetic DNA containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), we showed that p53 was pulled down together with two BER proteins, human 8-oxoguanine glycosylase (hOGG1) and AP endonuclease (APE). Functional analysis showed that p53 significantly enhanced the sequential activities of hOGG1 and APE in excising the 8-oxoG nucleotide from DNA in vitro. The ability of p53 to enhance the removal of oxidized DNA bases was further demonstrated in vivo using a pair of p53 isogenic lines. HCT116 p53+/+ cells exhibit a more rapid removal of 8-oxoG from DNA than p53-/- cells exposed to the same levels of reactive oxygen species (ROS) stress. Together, these results suggest that p53 participates in sensing oxidative DNA damage and modulates BER function in response to persistent ROS stress.
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PMID:Role of p53 in sensing oxidative DNA damage in response to reactive oxygen species-generating agents. 1534 9

The eukaryotic 8-oxoguanine-DNA glycosylase 1 (OGG1) provides the major activity for repairing mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) induced in the genome due to oxidative stress. Earlier in vitro studies showed that, after excising the base lesion, the human OGG1 remains bound to the resulting abasic (AP) site in DNA and does not turn over efficiently. The human AP-endonuclease (APE1), which cleaves the phosphodiester bond 5' to the AP site, in the next step of repair, displaces the bound OGG1 and thus increases its turnover. Here we show that NEIL1, a DNA glycosylase/AP lyase specific for many oxidized bases but with weak 8-oxoG excision activity, stimulates turnover of OGG1 in a fashion similar to that of APE1 and carries out betadelta-elimination at the AP site. This novel collaboration of two DNA glycosylases, which do not stably interact with each other, in stimulating 8-oxoguanine repair is possible because of higher AP site affinity and stronger AP lyase activity of NEIL1 relative to OGG1. Comparable levels of NEIL1 and OGG1 in some human cells raise the possibility that NEIL1 serves as a backup enzyme to APE1 in stimulating 8-oxoG repair in vivo.
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PMID:Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases. 1535 Jan 46

One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3'-blocking termini following AP lyase cleavage by Nth1.
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PMID:A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe. 1545 79


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