共查询到20条相似文献,搜索用时 0 毫秒
1.
2.
Apurinic and/or apyrimidinic (AP) sites were excised from PM2 phage DNA by two enzymes: an AP endodeoxyribonuclease isolated from rat neocortex chromatin and a rat brain exodeoxyribonuclease, DNase B III. The resulting gap was filled with DNA polymerase beta prepared from rat liver and finally ligated by Escherichia coli DNA ligase. 相似文献
3.
It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli. A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate. It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E. coli. The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM. It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione. The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases. 相似文献
4.
《Mutation Research/Genetic Toxicology》1990,240(2):93-100
Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV. 相似文献
5.
Mitochondrial endonuclease activities specific for apurinic/apyrimidinic sites in DNA from mouse cells 总被引:7,自引:0,他引:7
Endonuclease activity which specifically cleaves baseless (apurinic/apyrimidinic (AP] sites in supercoiled DNA has been purified from mitochondria of the mouse plasmacytoma cell line, MPC-11. Two variant forms separate upon purification; these have small but reproducible differences in catalytic and chromatographic properties, but similar physical properties. Both have a sedimentation coefficient of 4.0, corresponding to a molecular weight of 61,000 (assuming a globular configuration) and a peptide molecular weight of about 65,000 as determined by immunoblot analysis with antiserum raised against the major AP endonuclease from HeLa cells. Thus mitochondrial AP endonuclease appears to be a monomer of about 65 kDa, making it distinguishable from the major AP endonuclease of MPC-11 cells which, like those of other mammalian cells, appears to be a monomer of about 41 kDa. A possible 82-kDa precursor form was also detected by immunoblot analysis of a crude mitochondrial fraction. Mitochondrial AP endonuclease activity is greatly stimulated by divalent cations, has a pH optimum between 6.5 and 8.5, and cleaves the AP site by a class II mechanism to generate a 3'-OH nucleotide residue. These properties resemble those of the major mammalian AP endonucleases but, unlike those enzymes, mitochondrial AP endonuclease activity is neither inhibited by adenine or NAD+ nor stimulated by Triton X-100. Since the mitochondrial activity generates active primer termini for DNA synthesis, it could function in base excision DNA repair; alternatively, it might have a role in eliminating damaged mitochondrial genomes from the gene pool. 相似文献
6.
The excision repair of DNA damaged by physical or chemical agents may produce either apurinic/apyrimidinic (AP) sites or single-strand breaks (SSB) in the DNA. Alkaline-sucrose gradient sedimentation and alkaline elution, techniques generally used for the study of DNA repair which depend upon high pH to denature the DNA, cannot differentiate between these possibilities. A simple method for the quantitative measurement of SSB in DNA which leaves any AP sites intact is presented. This method relies upon the separation by size of the fragments resulting from the denaturation of the DNA under neutral conditions by sedimentation through gradients of sucrose in formamide. By combining the use of both formamide- and alkaline-sucrose sedimentation methods, we can quantify both AP sites and SSB in DNA. 相似文献
7.
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions. 相似文献
8.
9.
Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage. 总被引:4,自引:0,他引:4
Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV. 相似文献
10.
Mechanism of DNA strand nicking at apurinic/apyrimidinic sites by Escherichia coli [formamidopyrimidine]DNA glycosylase. 总被引:8,自引:0,他引:8
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Escherichia coli [formamidopyrimidine]DNA glycosylase catalyses the nicking of both the phosphodiester bonds 3' and 5' of apurinic or apyrimidinic sites in DNA so that the base-free deoxyribose is replaced by a gap limited by 3'-phosphate and 5'-phosphate ends. The two nickings are not the results of hydrolytic processes; the [formamidopyrimidine]DNA glycosylase rather catalyses a beta-elimination reaction that is immediately followed by a delta-elimination. The enzyme is without action on a 3'-terminal base-free deoxyribose or on a 3'-terminal base-free unsaturated sugar produced by a beta-elimination reaction nicking the DNA strand 3' to an apurinic or apyrimidinic site. 相似文献
11.
Delta-elimination in the repair of AP (apurinic/apyrimidinic) sites in DNA. 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
[5'-32P]pdT8d(-)dT7, containing an AP (apurinic/apyrimidinic) site in the ninth position, and [d(-)-1',2'-3H, 5'-32P]DNA, containing AP sites labelled with 3H in the 1' and 2' positions of the base-free deoxyribose [d(-)] and with 32P 5' to this deoxyribose, were used to investigate the yields of the beta-elimination and delta-elimination reactions catalysed by spermine, and also the yield of hydrolysis, by the 3'-phosphatase activity of T4 polynucleotide kinase, of the 3'-phosphate resulting from the beta delta-elimination. Phage-phi X174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: beta-elimination, delta-elimination, hydrolysis of 3'-phosphate, DNA polymerization and ligation. Spermine, in one experiment, and Escherichia coli formamidopyrimidine: DNA glycosylase, in another experiment, were used to catalyse the first and second steps (beta-elimination and delta-elimination). These repair pathways, involving a delta-elimination step, may be operational not only in E. coli repairing its DNA containing a formamido-pyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites. 相似文献
12.
Importance of thiols in the repair mechanisms of DNA containing AP (apurinic or apyrimidinic) sites. 总被引:5,自引:2,他引:3
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Addition of thiol compounds containing an anionic group to the 3'-terminal unsaturated sugar of the 5' fragment obtained from an oligonucleotide containing an AP site cleaved by beta-elimination, can be followed by gel electrophoresis. The technique enables to distinguish between two mechanisms of cleavage of the C3'-O-P bond 3' to an AP site: hydrolysis or beta-elimination. Addition of thiols to the double-bond of the 3'-terminal sugar resulting from beta-elimination prevents a subsequent delta-elimination. The interpretation of the action of enzymes that start by nicking 3' to AP sites must take into account the presence or absence of thiols in the reaction medium. In living cells, thiols might influence the pathways followed by the repair processes of AP site-containing DNA. 相似文献
13.
14.
Kutuzov MM Ilina ES Sukhanova MV Pyshnaya IA Pyshnyi DV Lavrik OI Khodyreva SN 《Biochemistry. Biokhimii?a》2011,76(1):147-156
To study the interaction of poly(ADP-ribose) polymerase 1 (PARP1) with apurinic/apyrimidinic sites (AP sites) within clustered
damages, DNA duplexes were created that contained an AP site in one strand and one of its analogs situated opposite the AP
site in the complementary strand. Residues of 3-hydroxy-2-hydroxymethyltetrahydrofuran (THF), diethylene glycol (DEG), and
decane-1,10-diol (DD) were used. It is shown for the first time that apurinic/apyrimidinic endonuclease 1 (APE1) cleaves the
DNA strands at the positions of DEG and DD residues, and this suggests these groups as AP site analogs. Insertion of DEG and
DD residues opposite an AP site decreased the rate of AP site hydrolysis by APE1 similarly to the effect of the THF residue,
which is a well-known analog of the AP site, and this allowed us to use such AP DNAs to imitate DNA with particular types
of clustered damages. PARP1, isolated and in cell extracts, efficiently interacted with AP DNA with analogs of AP sites producing
a Schiff base. PARP1 competes with APE1 upon interaction with AP DNAs, decreasing the level of its cross-linking with AP DNA,
and inhibits hydrolysis of AP sites within AP DNAs containing DEG and THF residues. Using glutaraldehyde as a linking agent,
APE1 is shown to considerably decrease the amount of AP DNA-bound PARP1 dimer, which is the catalytically active form of this
enzyme. Autopoly(ADP-ribosyl)ation of PARP1 decreased its inhibitory effect. The possible involvement of PARP1 and its automodification
in the regulation of AP site processing within particular clustered damages is discussed. 相似文献
15.
Two distinct endonucleases from Saccharomyces cerevisiae, specific for apurinic/apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from unspecific and ultraviolet-specific endonucleases and exonucleases. The two enzymes are monomeric proteins of around 24000 daltons. Both are sensitive to ionic strength and most active in the presence of 150 and 100 mM NaCl for AP-endonucleases A and B, respectively. They are not absolutely dependent on divalent cations, since they are insensitive to EDTA, although AP-endonuclease A is activated by Ca2+ or Mg2+ and AP-endonuclease B by Mg2+ only. ATP inhibits the enzymes. AP-endonuclease A reacts optimally between pH 6 and 8, and AP-endonucleases B at pH 8. AP-endonuclease A is more stable at 60 degree C (half-life of 17 min) than B (half-life of 4 min). AP-endonuclease A is insensitive to N-ethylmaleimide or rho-chloromercuribenzoate. AP-endonuclease B is also insensitive to N-ethylmaleimide, but rho-chloromercuribenzoate inhibits its activity. 相似文献
16.
Alexander T. Akhmedov Oleg K. Kaboev Michael L. Bekker 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,696(2):163-170
Two distinct endonucleases from Saccharomyces cerevisiae, specific for apurinic/apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from unspecific and ultraviolet-specific endonucleases and exonucleases. The two enzymes are monomeric proteins of around 24 000 daltons. Both are sensitive to ionic strength and most active in the presence of 150 and 100 mM NaCl for AP-endonucleases A and B, respectively. They are not absolutely dependent on divalent cations, since they are insensitive to EDTA, although AP-endonuclease A is activated by Ca2+ or Mg2+ and AP-endonuclease B by Mg2+ only. ATP inhibits the enzymes. AP-endonuclease A reacts optimally between pH 6 and 8, and AP-endonuclease B at pH 8. AP-endonuclease A is more stable at 60°C (half-life of 17 min) than B (half-life of 4 min). AP-endonucleuase A is insensitive to N-ethylmaleimide or ρ-chloromercuribenzoate. AP-endonuclease B is also insensitive to N-ethylmaleimide, but ρ-chloromercuribenzoate inhibits its activity. 相似文献
17.
18.
Differential reactivity of 9-NH2-ellipticine on apurinic and apyrimidinic sites in circular DNA 总被引:1,自引:0,他引:1
Endonucleases for apurinic sites as well as chemical compounds reacting with aldehydes do not generally differentiate between apurinic and apyrimidinic sites. We have studied the effect of the apurinic site reagent, 9-NH2-ellipticine, on apyrimidinic sites enzymatically generated on PBR322 DNA and compared it to its' action on apurinic PM2 and PBR322 DNAs. In conditions where this compound induces breakage of apurinic sites, it does not display any action on apyrimidinic sites. 相似文献
19.
Apurinic/apyrimidinic endonuclease (AP endo) is believed to play a critical role in repair of oxidative damage of DNA and is proposed to initiate repair of most abasic sites in the base excision repair pathway. AP endo makes a single nick 5' to an abasic site in double-stranded DNA. In this study, we investigated whether AP endo locates an abasic site through a processive or a distributive mechanism. We used a linear multi-abasic site substrate (concatemer), synthesized by ligating together identical 25-nucleotide monomeric units (25-mers). We first determined that the 25-mer monomer from which the concatemers were prepared was nicked by AP endo in a fashion similar to that of the previously published 49-mer substrate with a different sequence. Steady state parameters K(m) and k(cat) and single-turnover parameters for substrate binding were comparable to previously published values. Using the multi-abasic site concatemer, we demonstrated that AP endo was capable of cleaving approximately seven to eight abasic sites, traveling at least 200 nucleotides, before dissociating from its substrate. Thus, AP endo, like uracil DNA glycosylase, behaves in a quasi processive fashion. Processivity could be separated from catalysis, since processivity was maximal at 25 mM NaCl, while the rate of cleavage was maximal at 125 mM salt. In short, nicking activity was maximized close to physiological salt molarities while processivity was midrange at physiological salt concentrations. The latter is likely to be subject to tight regulation by small changes in ionic strength. 相似文献
20.
Human apurinic/apyrimidinic endonuclease 1 (APE1) is one of the key participants in the DNA base excision repair system. APE1 hydrolyzes DNA adjacent to the 5′-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3′-hydroxyl group and a 5′-deoxyribose phosphate moiety. APE1 exhibits 3′-phosphodiesterase, 3′-5′-exonuclease, and 3-phosphatase activities. APE1 was also identified as a redox factor (Ref-1). In this review, data on the role of APE1 in the DNA repair process and in other metabolic processes occurring in cells are analyzed as well as the interaction of this enzyme with DNA and other proteins participating in the repair system. 相似文献