共查询到20条相似文献,搜索用时 62 毫秒
1.
gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed. 相似文献
2.
Povirk LF Zhou T Zhou R Cowan MJ Yannone SM 《The Journal of biological chemistry》2007,282(6):3547-3558
The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3'-hydroxyl or 3'-phosphoglycolate moieties, was examined. A 3'-phosphoglycolate had little effect on Artemis-mediated trimming of long 3' overhangs (> or =9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3'-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3'-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3' overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3' overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3' overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3' to the cleavage site, as well as 2 unpaired nucleotides 5' to the cleavage site. Shorter 3'-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3'-phosphoglycolate-terminated double strand breaks. 相似文献
3.
DNA strand breaks produced by oxidative stress in mammalian cells exhibit 3'-phosphoglycolate termini.
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)
In recent years two mechanisms have been proposed for the production of DNA strand breaks in cells undergoing oxidative stress: (i) DNA attack by OH radical, produced by Fenton reaction catalyzed by DNA-bound iron; and (ii) DNA attack by calcium-activated nucleases, due to the increase of cytosolic and nuclear calcium induced by oxidative stress. We set out to investigate the participation of the former mechanism by detecting and quantifying 3'-phosphoglycolate, a 3' DNA terminus known to be formed by OH radical attack to the deoxyribose moiety, followed by sugar ring rupture and DNA strand rupture. These structures were found in DNA of monkey kidney cells exposed to hydrogen peroxide, iron nitrilotriacetate or ascorbate, all species known to favor a cellular pro-oxidant status. The method employed to measure 3' phosphoglycolate was the 32P-postlabeling assay. Repair time course experiments showed that it takes 10 h for 3'-phosphoglycolate to be removed from DNA. It was found that the DNA of both control cells and cells exposed to hydrogen peroxide had a very poor capacity of supporting in vitro DNA synthesis, catalyzed by DNA polymerase I. If the DNA was previously incubated with exonuclease III, an enzyme able to expose 3'-OH primers by removal of 3'-phosphoglycolate and 3'-phosphate termini the in vitro synthesis was substantially increased. This result shows that either of these termini are present at the break and that 3'-hydroxyl termini are virtually absent. At least 25% of the strand breaks exhibited 3'-phosphoglycolate termini as determined by the 32P-postlabeling assay, but due to the characteristic of the method this percentage is likely to be higher. These results favor the hypothesis that an oxidative agent generated by Fenton reaction is responsible for DNA strand breakage in cells undergoing oxidative stress. 相似文献
4.
We have purified three chromatographically distinct human enzyme activities from HeLa cells, that are capable of converting bleomycin-treated DNA into a substrate for E. coli DNA polymerase I. The bleomycin-treated DNA substrate used in this study has been characterized via a 32P-postlabeling assay and shown to contain strand breaks with 3'-phosphoglycolate termini as greater than 95% of the detectable dose-dependent lesions. The purified HeLa cell enzymes were shown to be capable of removing 3'-phosphoglycolates from this substrate. Also 3'-phosphoglycolate removal and nucleotide incorporation were enzyme dependent. In addition, all three Hela cell enzymes have been determined to possess Class II AP endonuclease activity. The enzymes lack 3'----5' exonuclease activity and are, therefore, dissimilar to exonuclease III--an E. coli enzyme that can remove 3'-phosphoglycolate. 相似文献
5.
Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1. 总被引:4,自引:2,他引:2
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)
T A Winters W D Henner P S Russell A McCullough T J Jorgensen 《Nucleic acids research》1994,22(10):1866-1873
A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries. This oligodeoxyribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation. HAP1 was found to recognize the strand break, and catalyze the release of the 3'-phosphoglycolate as free phosphoglycolic acid. The enzyme had a Vmax of 0.1 fmole/min/pg of HAP1 protein, and a Km of 0.05 microM for the 3'-phosphoglycolate strand break lesion. The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3'-phosphoglycolate moiety and the 3'-carbon of the adjacent dGMP moiety within the oligonucleotide. The resulting DNA contained a 3'-hydroxyl which supported nucleotide incorporation by E. coli DNA polymerase I large fragment. AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site. The Vmax and Km for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 microM, respectively. 相似文献
6.
Human tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety. In eukaryotic cells, this type of linkage is found in stalled topoisomerase I-DNA covalent complexes, and Tdp1 has been implicated in the repair of such complexes in vivo. We confirm here that the Tdp1 catalytic cycle involves a covalent reaction intermediate in which a histidine residue is connected to a DNA 3'-phosphate through a phosphoamide linkage. Most surprisingly, this linkage can be hydrolyzed by Tdp1, and unlike a topoisomerase I-DNA complex, which requires modification to be an efficient substrate for Tdp1, the native form of Tdp1 can be removed from the DNA. The spinocerebellar ataxia with axonal neuropathy neurodegenerative disease is caused by the H493R mutant form of Tdp1, which shows reduced enzymatic activity and accumulates the Tdp1-DNA covalent intermediate. The ability of wild type Tdp1 to remove the stalled mutant protein from the DNA likely explains the recessive nature of spinocerebellar ataxia with axonal neuropathy. In addition to its activity on phosphotyrosine and phosphohistidine substrates, Tdp1 also possesses a limited DNA and RNA 3'-exonuclease activity in which a single nucleoside is removed from the 3'-hydroxyl end of the substrate. Furthermore, Tdp1 also removes a 3' abasic site and an artificial 3'-biotin adduct from the DNA. In combination with earlier data showing that Tdp1 can use 3'-phosphoglycolate as a substrate, these data suggest that Tdp1 may function to remove a variety of 3' adducts from DNA during DNA repair. 相似文献
7.
Inamdar KV Pouliot JJ Zhou T Lees-Miller SP Rasouli-Nia A Povirk LF 《The Journal of biological chemistry》2002,277(30):27162-27168
Mammalian cells contain potent activity for removal of 3'-phosphoglycolates from single-stranded oligomers and from 3' overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a single-stranded 3' terminus to leave a 3'-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3'-phosphoglycolate on a 3-base 3' overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3' overhangs. 相似文献
8.
Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase alpha. Purification and properties of the exonuclease 总被引:7,自引:0,他引:7
The DNase that is associated with a multiprotein form of HeLa cell DNA polymerase alpha (polymerase alpha 2) has two distinct exonuclease activities: the major activity initiates hydrolysis from the 3' terminus and the other from the 5' terminus of single-stranded DNA. The two exonuclease activities show identical rates of thermal inactivation and coincidental migration during chromatofocusing, glycerol gradient centrifugation, and nondenaturing polyacrylamide gel electrophoresis of the DNase. Moreover, the purified DNase shows a single protein band of Mr 69,000 following nondenaturing polyacrylamide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 3'----5' exonuclease activity hydrolyzes only single-stranded DNA substrates and the products are 5' mononucleotides. This activity recognizes and excizes mismatched bases at the 3' terminus of double-stranded DNA substrates. The 3'----5' exonuclease does not hydrolyze 3' phosphoryl terminated single-stranded DNA substrates. The 5'----3' exonuclease activity also only hydrolyzes single-stranded DNA substrates. The rate of hydrolysis, however is only about 1/25th the rate of the 3'----5' exonuclease. This exonuclease activity requires a 5' single-stranded terminus in order to initiate hydrolysis and does not proceed into double-stranded regions. The products of hydrolysis by 5'----3' exonuclease are also 5' nucleoside monophosphates. 相似文献
9.
An endonuclease activity has been purified approximately 800-fold from nuclei of 3T3 cells infected with polyoma virus. The purfied enzyme catalyzes an endonucleoytic cleavage of single- and double-stranded DNA and single-stranded RNA. Evidence that the activity towards these substrates resides in the same protein molecule is provided by the finding that they co-sediment in sucrose gradients and have identical rates of heat inactivation. Studies on the DNase activity shows that the rate of hydrolysis of single-stranded T7 DNA is 100-fold greater than that for double-stranded T7 DNA. Single-stranded DNA is extensively hydrolyzed to low molecular weight acid-insoluble products. With duplex DNA as substrate, only a limited number of single strand breaks are introduced. A limit digest with polyoma DNA (component I) as substrate results in the introduction of four breaks per strand. The phosphdiester bond interruptions can be repaired by polynucleotide ligase. Approximately 80% of the 5' termini present at the point of phosphodiester bond cleavage are purine nucleotides. Additional studies have demonstrated that a similar endonuclease is present in nuclei of uninfected cells and that this enzyme purified 400-fold has catalytic properties identical with those of the endonuclease from infected cells. 相似文献
10.
Chen S Hannis JC Flora JW Muddiman DC Charles K Yu Y Povirk LF 《Analytical biochemistry》2001,289(2):274-280
Single- and double-strand breaks bearing 3'-phosphoglycolate termini are among the most frequent lesions formed in DNA by ionizing radiation and other oxidative mutagens. In order to obtain homogeneous preparations of defined 3'-phosphoglycolate substrates for repair studies, 5'-(32)P-end-labeled partial duplex DNAs were treated with bleomycin, and individual cleavage products were isolated from polyacrylamide gels. The fragments were then treated with alkaline phosphatase and further purified by reverse-phase HPLC. Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry of the purified oligomers produced molecular ions of the expected masses, with no detectable contaminants. Gas-phase sequencing by tandem mass spectrometry of these single species yielded the expected sequence ions and confirmed the presence of phosphoglycolate on the 3'-terminal fragments only. The fragments could be relabeled with polynucleotide kinase to yield highly purified, high-specific-activity substrates for repair studies. 相似文献
11.
Wilson DM 《Journal of molecular biology》2003,330(5):1027-1037
Ape1 is the major human abasic endonuclease, initiating repair of this common DNA lesion by incising the phosphodiester backbone 5' to the damage site. This enzyme also functions in specific contexts to excise 3'-blocking termini, e.g. phosphate and phosphoglycolate residues, from DNA. Recently, the comparatively "minor" 3' to 5' exonuclease activity of Ape1 was found to contribute to the excision of certain 3'-mismatched nucleotides. In this study, I characterize more thoroughly the 3'-nuclease properties of Ape1 and define the effects of specific DNA determinants on this function. Data within shows that Ape1 is a non- or poorly processive exonuclease, which degrades one nucleotide gap, 3'-recessed, and nicked DNAs, but exhibits no detectable activity on blunt end or single-stranded DNA. A 5'-phosphate, compared to a 5'-hydroxyl group, reduced Ape1 degradation activity roughly tenfold, suggesting that the biological impact of certain DNA single strand breaks may be influenced by the terminal chemistry. In the context of a base excision repair-like DNA intermediate, a 5'-abasic residue exerted an about tenfold attenuation on the 3' to 5' exonuclease efficiency of Ape1. A 3'-phosphate group had little impact on Ape1 exonuclease activity, and oligonucleotides harboring these blocking termini were activated by Ape1 for DNA polymerase beta extension. Ape1 was also found to remove 3'-tyrosyl residues from 3'-recessed and nicked DNAs, suggesting a potential role in processing covalent topoisomerase I-DNA intermediates formed during chromosome relaxation. While exhibiting preferential excision of thymine in a T:G mismatch context, Ape1 was unable to degrade a triple 3'-thymine mispair. However, Ape1 was able to excise double nucleotide mispairs, apparently through a novel 3'-flap-type endonuclease activity, again activating these substrates for polymerase beta extension. 相似文献
12.
Chen S Inamdar KV Pfeiffer P Feldmann E Hannah MF Yu Y Lee JW Zhou T Lees-Miller SP Povirk LF 《The Journal of biological chemistry》2001,276(26):24323-24330
To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3'-overhangs and a one-base gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3' --> 5' resection before ligation, was formed. Similar results were seen for a substrate with 5'-overhangs and recessed 3'-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation. 相似文献
13.
Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen. 总被引:22,自引:4,他引:22
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
N Tuteja R Tuteja A Ochem P Taneja N W Huang A Simoncsits S Susic K Rahman L Marusic J Chen et al. 《The EMBO journal》1994,13(20):4991-5001
Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3' to 5' direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single-stranded DNA-dependent ATPase activity, as well as double-stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha-32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross-reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA-dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double-stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism. 相似文献
14.
Ionizing radiation, oxidative stress and endogenous DNA-damage processing can result in a variety of single-strand breaks with modified 5' and/or 3' ends. These are thought to be one of the most persistent forms of DNA damage and may threaten cell survival. This study addresses the mechanism involved in recognition and processing of DNA strand breaks containing modified 3' ends. Using a DNA-protein cross-linking assay, we followed the proteins involved in the repair of oligonucleotide duplexes containing strand breaks with a phosphate or phosphoglycolate group at the 3' end. We found that, in human whole cell extracts, end-damage-specific proteins (apurinic/apyrimidinic endonuclease 1 and polynucleotide kinase in the case of 3' ends containing phosphoglycolate and phosphate, respectively) which recognize and process 3'-end-modified DNA strand breaks are required for efficient recruitment of X-ray cross-complementing protein 1-DNA ligase IIIalpha heterodimer to the sites of DNA repair. 相似文献
15.
3'-phosphodiesterase and 3'-->5' exonuclease activities of yeast Apn2 protein and requirement of these activities for repair of oxidative DNA damage
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
In Saccharomyces cerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites. Oxidative DNA-damaging agents, such as H(2)O(2), produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. Here, we show that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'-->5' exonuclease activities, and mutation of the active-site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, genetic studies indicate the involvement of APN2 in the repair of H(2)O(2)-induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we conclude that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species. 相似文献
16.
End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3'-end groups of strand breaks caused by gamma radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3'-phosphoglycolate and 3'-phosphate, with 5'-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3'-hydroxyl (3'-OH) and 3'-phosphate (3'-P) end groups and 5'-ends (> or = 42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the 125I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer. 相似文献
17.
Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and Werner's syndrome genes. We have studied the substrate specificity of a recombinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein). Sgs1 shows a strong preference for binding branched DNA substrates, including duplex structures with a 3' single-stranded overhang and DNA junctions with multiple branches. Duplex DNA with a 5' rather than a 3' single-stranded tail is not recognized or unwound by Sgs1. DNase I and hydroxyl radical footprinting of the Sgs1-DNA complex shows that the protein binds specifically to the junction of a double-stranded DNA and its 3' overhang. Binding and unwinding of duplex DNA with a 3' overhang are much reduced if the backbone polarity of the 3' overhang is reversed in the junction region, but are unaffected if polarity reversal occurs four nucleotides away from the junction. These results indicate that the 3' to 5' polarity of unwinding by the recombinant Sgs1 protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinant Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo. 相似文献
18.
Opresko PL Laine JP Brosh RM Seidman MM Bohr VA 《The Journal of biological chemistry》2001,276(48):44677-44687
Werner syndrome is a human disorder characterized by premature aging, genomic instability, and abnormal telomere metabolism. The Werner syndrome protein (WRN) is the only known member of the RecQ DNA helicase family that contains a 3' --> 5'-exonuclease. However, it is not known whether both activities coordinate in a biological pathway. Here, we describe DNA structures, forked duplexes containing telomeric repeats, that are substrates for the simultaneous action of both WRN activities. We used these substrates to study the interactions between the WRN helicase and exonuclease on a single DNA molecule. WRN helicase unwinds at the forked end of the substrate, whereas the WRN exonuclease acts at the blunt end. Progression of the WRN exonuclease is inhibited by the action of WRN helicase converting duplex DNA to single strand DNA on forks of various duplex lengths. The WRN helicase and exonuclease act in concert to remove a DNA strand from a long forked duplex that is not completely unwound by the helicase. We analyzed the simultaneous action of WRN activities on the long forked duplex in the presence of the WRN protein partners, replication protein A (RPA), and the Ku70/80 heterodimer. RPA stimulated the WRN helicase, whereas Ku stimulated the WRN exonuclease. In the presence of both RPA and Ku, the WRN helicase activity dominated the exonuclease activity. 相似文献
19.
The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides. 相似文献
20.
Mechanism of interaction between Ku protein and DNA 总被引:60,自引:0,他引:60
The mechanism of interaction between the Ku autoantigenic protein, a heterodimer of noncovalently linked 70,000- and 80,000-dalton subunits, and DNA was studied using immunoaffinity-purified Ku protein and a 300-base pair EcoRI fragment from HeLa cell DNA. In the nitrocellulose filter-binding assay, the Ku protein bound 32P-labeled double-stranded DNA, and much less efficiently single-stranded DNA. The binding of Ku to DNA was dependent on ionic strength and prevented by IgG from patient sera containing anti-Ku antibodies. In competitive assays, using unlabeled nucleic acid competitors, the DNA binding of Ku was not inhibited in the presence of yeast tRNA, synthetic copolymer of poly(A)-poly(dT), or circular plasmid pBR322 DNA, but was inhibited when the plasmid DNA was cleaved with appropriate restriction endonucleases. The inhibitory activities of cleaved plasmid DNA were independent of the configuration or nucleotide sequences at ends but proportional to the number of recognition sites of restriction enzymes used. Footprint analysis demonstrated that Ku protein protected both 3'- and 5'-terminal regions of double-stranded DNA from DNase I digestion. When Ku protein was fractionated electrophoretically, transferred to nitrocellulose filter, and probed with 32P-labeled DNA, only the 70,000-dalton subunit exhibited DNA binding. Thus, the Ku protein appears to recognize selectively ends of double-stranded DNA molecules. Possible functions of the Ku autoantigen in eukaryotic cells are discussed. 相似文献