首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Sung JS  Mosbaugh DW 《Biochemistry》2003,42(16):4613-4625
The rate, extent, and DNA synthesis patch size of base excision repair (BER) were measured using Escherichia coli GM31 cell-free extracts and a pGEM (form I) DNA substrate containing a site-specific uracil or ethenocytosine target. The rate of complete BER was stimulated (approximately 3-fold) by adding exogenous E. coli DNA ligase to the cell-free extract, whereas addition of E. coli Ung, Nfo, Fpg, or Pol I did not stimulate BER. Hence, DNA ligation was identified as the rate-limiting step in the E. coli BER pathway. The addition of exogenous DNA polymerase I caused modest inhibition of BER, which was overcome by concomitant addition of DNA ligase. Repair patch size determinations were performed to assess the distribution of DNA synthesis associated with both uracil- and ethenocytosine-initiated BER. During the early phase (0-5 min) of the BER reaction, the large majority of repair events resulted from short patch (1-nucleotide) DNA synthesis. However, during the late phase (>10 min) both short and long (2-20 nucleotide) patches were observed, with long patch BER progressively dominating the repair process. In addition, the patch size distribution was influenced by the ratio of DNA polymerase I to DNA ligase activity in the reaction. A novel mode of BER was identified that involved DNA synthesis tracts of >205 nucleotides in length and termed very-long patch BER. This BER process was dependent upon DNA polymerase I since very-long patch BER was inhibited by DNA polymerase I antibody and addition of excess DNA polymerase I reversed this inhibition.  相似文献   

2.
Bacteria and eukaryotic cells employ a variety of enzymatic pathways to remove damage from DNA or to lessen its impact upon cellular functions. Most of these processes were discovered in Escherichia coli and have been most extensively analyzed in this organism because suitable mutants have been isolated and characterized. Analogous pathways have been inferred to exist in mammalian cells from the presence of enzyme activities similar to those known to be involved in repair in bacteria, from the analysis of events in cells treated with DNA damaging agents, and from the analysis of the few naturally occurring mutant cell types. Excision repair of pyrimidine dimers produced by UV in E coli is initiated by an incision event catalyzed by a complex composed of uvrA, uvrB, and uvrC gene products. Multiple exonuclease and polymerase activities are available for the subsequent excision and resynthesis steps. In addition to the constitutive pathway, which produces short patches of 20–30 nucleotides, an inducible excision repair process exists that produces much longer patches. This long patch pathway is controlled by the recA-lexA regulatory circuit and also requires the recF gene. It is apparently not responsible for UV-induced mutagenesis. However, the ability to perform inducible long patch repair correlates with enhanced bacterial survival and with a major component of the Weigle reactivation of bacteriophage with double-strand DNA genomes. Mammalian cells possess an excision repair pathway similar to the constitutive pathway in E coli. Although not as well understood, the incision event is at least as complex, and repair resynthesis produces patches of about the same size as the constitutive short patches. In mammalian cells, no patches comparable in size to those produced by the inducible pathway of E coli are observed. Repair in mammalian cells may be more complicated than in bacteria because of the structure of chromatin, which can affect both the distribution of DNA damage and its accessibility to repair enzymes. A coordinated alteration and reassembly of chromatin at sites of repair may be required. We have observed that the sensitivity of digestion by staphylococcal nuclease (SN) of newly synthesized repair patches resulting from excision of furocoumarin adducts changes with time in the same way as that of patches resulting from excision of pyrimidine dimers. Since furocoumarin adducts are formed only in the SN-sensitive linker DNA between nucleosome cores, this suggests that after repair resynthesis is completed, the nucleosome cores in the region of the repair event do not return exactly to their original positions. We have also studied excision repair of UV and chemical damage in the highly repeated 172 base pair α DNA sequence in African green monkey cells. In UV irradiated cells, the rate and extent of repair resynthesis in this sequence is similar to that in bulk DNA. However, in cells containing furocoumarin adducts, repair resynthesis in α DNA is only about 30% of that in bulk DNA. Since the frequency of adducts does not seem to be reduced in α DNA, it appears that certain adducts in this unique DNA may be less accessible to repair. Endonuclease V of bacteriophage T4 incises DNA at pyrimidine dimers by cleaving first the glycosylic bond between deoxyribose and the 5′ pyrimidine of the dimer and then the phosphodiester bond between the two pyrimidines. We have cloned the gene (denV) that codes for this enzyme and have demonstrated its expression in uvrA recA and uvrB recA cells of E coli. Because T4 endonuclease V can alleviate the excision repair deficiency of xeroderma pigmentosum when added to permeabilized cells or to isolated nuclei after UV irradiation, the cloned denV gene may ultimately be of value for analyzing DNA repair pathways in cultured human cells.  相似文献   

3.
Exposure of human cells to ionizing radiation at the G1/S-phase border of the cell cycle leads to the production of repair patches of 3 nucleotides, representing the constitutive repair response, and very long repair patches (VLRP) of at least 150 nucleotides, representing an induced response. We examined the type of DNA damage that may signal this induced repair response using two chemicals that produce subsets of the damage induced by ionizing radiation. Treatment of cells at the G1/S-phase border with bleomycin, which produces a high proportion of DNA double-strand breaks, also leads to the production of VLRP of at least 130 nucleotides. In contrast, when cells were treated with hydrogen peroxide, which produces base modifications and single-strand breaks, no VLRP were observed. Thus it would appear that DNA double-strand breaks are the signal that leads to the induction of the VLRP. We also examined the relationship between the induced repair response and DNA replication. When cells are treated with hydroxyurea, under conditions that inhibit more than 98% of the DNA synthesis, prior to exposure to 5 Gy, repair patches of 3 and 150 nucleotides are found. This indicates that the longer repair patches are not a result of aberrant DNA replication. However, when cells are treated with the DNA polymerase inhibitor aphidicolin in combination with hydroxyurea and cytosine arabinoside, no induced long patches are found. These results indicate that DNA polymerase alpha, delta or epsilon is required for the synthesis of the VLRP.  相似文献   

4.
    
Summary The ultraviolet (UV) sensitivity ofEscherichia coli mutants deficient in the 5′→3′ exonuclease activity of DNA polymerase I is intermediate between that ofpol + strains and mutants which are deficient in the polymerizing activity of pol I (polA1). LikepolA1 mutants, the 5′-exonuclease deficient mutants exhibit increased UV-induced DNA degradation and increased repair synthesis compared to apol + strain, although the increase is not as great as inpolA1 or in the conditionally lethal mutant BT4113ts deficient inboth polymerase I activities. When dimer excision was measured at UV doses low enough to avoid interference from extensive DNA degradation, all three classes of polymerase I deficient mutants were found to remove dimers efficiently from their DNA. We conclude that enzymes alternative to polymerase I can operate in both the excision and resynthesis steps of excision repair and that substitution for either of the polymerase I functions results in longer patches of repair. A model is proposed detailing the possible events in the alternative pathways.  相似文献   

5.
The lengths of ultraviolet irradiation-induced repair resynthesis patches were measured in repair-competent extracts of Escherichia coli. Extracts containing wild-type deoxyribonucleic acid (DNA) polymerase I introduced a patch 15 to 20 nucleotides in length during repair of ColE1 plasmid DNA; extracts containing the polA5 mutant form of DNA polymerase I introduced a patch only about 5 nucleotides in length in a similar reaction. The repair patch length in the presence of either DNA polymerase corresponded to the processivity of that polymerase (the average number of nucleotides added per enzyme-DNA binding event) as determined with purified enzymes and DNA treated with a nonspecific endonuclease. The base composition of the repair patch inserted by the wild-type DNA polymerase was similar to that of the bacterial genome, whereas the patch inserted by the mutant enzyme was skewed toward greater pyrimidine incorporation. This skewing is expected, considering the predominance of pyrimidine incorporation occurring at the ultraviolet lesion and the short patch made by the mutant enzyme. Since the defect in the polA5 DNA polymerase which causes premature dissociation from DNA is reflected exactly in the repair patch length, the processive mechanism of the polymerase must be a central determinant of patch length.  相似文献   

6.
Treatment of permeable human fibroblasts with bleomycin elicits DNA repair synthesis that is only partially sensitive to aphidicolin, an inhibitor of mammalian DNA polymerases alpha and delta. Inhibition of long-patch repair synthesis by omission of the three unlabeled deoxyribonucleoside triphosphates (dNTPs) selectively eliminates the aphidicolin-sensitive component. The majority of this residual aphidicolin-resistant repair synthesis is contained in ligated patches as revealed by resistance to exonuclease III. Determination of repair patch length by bromodeoxyuridine-induced density shift under conditions where essentially all of the repair synthesis is sensitive or resistant to aphidicolin yielded values of approximately 20 and 4 nucleotides per patch, respectively. On the basis of these data and the relative sensitivity of bleomycin-induced repair synthesis to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), and N-ethylmaleimide (NEM), long-patch repair is attributed to DNA polymerase delta and short-patch repair to DNA polymerase beta.  相似文献   

7.
Among the different base excision repair pathways known, the long patch base excision repair of apurinic/apyrimidinic sites is an important mechanism that requires proliferating cell nuclear antigen. We have reconstituted this pathway using purified human proteins. Our data indicated that efficient repair is dependent on six components including AP endonuclease, replication factor C, proliferating cell nuclear antigen, DNA polymerases delta or epsilon, flap endonuclease 1, and DNA ligase I. Fine mapping of the nucleotide replacement events showed that repair patches extended up to a maximum of 10 nucleotides 3' to the lesion. However, almost 70% of the repair synthesis was confined to 2-4-nucleotide patches and DNA ligase I appeared to be responsible for limiting the repair patch length. Moreover, both proliferating cell nuclear antigen and flap endonuclease 1 are required for the production and ligation of long patch repair intermediates suggesting an important role of this complex in both excision and resynthesis steps.  相似文献   

8.
Excision repair of DNA in the presence of aphidicolin   总被引:2,自引:0,他引:2  
During excision repair of UV light or dimethyl sulphate (DMS)-induced damage to DNA the patch size for actively replicating KB or T98G cells is around 20 nucleotides. When confluent T98G cells or 'quiescent' KB cells are used the patch size is around 10 nucleotides. This value can be increased to around 20 nucleotides in T98G cells if a large excess of BrdUrd is included in the repair incubation medium. With 'quiescent' KB cells the patch size is not increased by excess BrdUrd. For all of these experimental conditions, when excision repair of UV or DMS damage takes place in the presence of aphidicolin, the patch size is found to be several times that found in its absence. Given the inhibitory specificity of aphidicolin for DNA polymerase alpha these results provide additional evidence that DNA polymerase alpha plays a role in the excision repair of DNA damaged by UV light or DMS. It is postulated that aphidicolin interrupts the processivity of the DNA polymerase alpha holoenzyme and allows an exonuclease to enlarge the repair site.  相似文献   

9.
Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of -[32P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3 side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.  相似文献   

10.
Background: The base excision–repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision–repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision–repair, we have attempted to reconstitute the pathway in vitro using purified enzymes.Results We report here the successful reconstitution of the base excision–repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5′ terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5′ nuclease function of DNA polymerase I.Conclusion Repair of uracil in DNA is achieved by a very short-patch excision–repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.  相似文献   

11.
The bromodeoxyuridine density-shift technique was used to examine nucleotide and base DNA excision repair in quiescent and lectin stimulated bovine lymphocytes damaged with either ultraviolet light or dimethyl sulfate (DMS). Compared to a number of human cell lines, quiescent lymphocytes were less proficient in the repair of both types of damage. Repair replication was enhanced upon mitogenic stimulation, but both the amount and time course of the increase in repair depended upon the damaging agent used. A 2-3-fold increase in UV light induced repair replication occurred early during stimulation and subsided only gradually as stimulation proceeded. However, the profile of DMS induced repair increased 7-fold and then decreased, in parallel with measurements of lectin-stimulated DNA replication. Estimates of average repair patch sizes showed that quiescent lymphocytes produced smaller patches of 7 nucleotides in response to DMS damage while UV light irradiation resulted in repair patches of 20 nucleotides. During stimulation, patch sizes appeared to increase to maximum values of 45 and 33 nucleotides in response to UV light and DMS, respectively, one day prior to the peak of DNA replication. These increases in patch size were followed by a gradual decrease towards unstimulated levels. However, the appearance of a DNA species of intermediate density in the gradient profiles made the interpretation of repair patch sizes in stimulated cells difficult. These results are discussed as evidence not only for differences in the mechanisms of nucleotide and base excision repair but also for changes in repair as the cell progresses through the cell cycle.  相似文献   

12.
The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N6-ethenoadenine (epsilonA) are both substrates for the monofunctional 3-methyladenine DNA glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA glycosylase/beta-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, epsilonA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and epsilonA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase beta-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template.  相似文献   

13.
Excision repair and patch size in UV-irradiated bacteriophage T4.   总被引:3,自引:2,他引:1       下载免费PDF全文
We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.  相似文献   

14.
An apurinic/apyrimidinic (AP) site is one of the most abundant lesions spontaneously generated in living cells and is also a reaction intermediate in base excision repair. In higher eukaryotes, there are two alternative pathways for base excision repair: a DNA polymerase beta-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Here we have reconstituted PCNA-dependent repair of AP sites with six purified human proteins: AP endonuclease, replication factor C, PCNA, flap endonuclease 1 (FEN1), DNA polymerase delta, and DNA ligase I. The length of nucleotides replaced during the repair reaction (patch size) was predominantly two nucleotides, although longer patches of up to seven nucleotides could be detected. Neither replication protein A nor Ku70/80 enhanced the repair activity in this system. Disruption of the PCNA-binding site of either FEN1 or DNA ligase I significantly reduced efficiency of AP site repair but did not affect repair patch size.  相似文献   

15.
Petermann E  Keil C  Oei SL 《DNA Repair》2006,5(5):544-555
Damaged DNA bases are repaired by base excision repair (BER), which can proceed via two pathways: short patch and long patch BER. During the latter, a stretch of several nucleotides is replaced by strand displacement DNA synthesis. We recently demonstrated that the ATP concentration may govern the decision between these BER sub-pathways. Employing a reconstituted BER complex containing among others DNA polymerase beta (Pol beta), DNA ligase III (Lig III) and XRCC1, here we show that Lig III and XRCC1 are essential mediators of this regulation. XRCC1 stimulates Pol beta strand displacement activity and releases inhibition of Pol beta by DNA-bound Lig III if ligation is prevented. XRCC1 is thus able to strongly promote strand displacement and long patch BER under conditions of ATP shortage. If sufficient ATP is available, ligation by Lig III prevents strand displacement, leading to short patch BER. Ligation-inactive mutants of Lig III do not prevent strand displacement by Pol beta under the same conditions. Consequently, the preferred use of short patch BER depends on the ligation competence of Lig III. Accordingly, lowering the levels of the XRCC1/Lig III complex in HeLa cells using siRNA decreases ligation capacity but enhances Pol beta-dependent DNA synthesis.  相似文献   

16.
Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of α-[32P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3′ side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.  相似文献   

17.
We have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells. DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 degrees C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 degrees C. Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred. Thus the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating step(s) of DNA repair. DNA repair patches synthesized in UV-irradiated cells labeled at 37 degrees C with [3H]Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA. DNA repair patches synthesized at 45 degrees C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures.  相似文献   

18.
We have previously described a novel DNA repair response that is induced in cells irradiated with ionizing radiation at the G1/S-phase border and is characterized by the formation of very long repair patches (VLRP) containing at least 150 nucleotides. In the current study, we examined whether there is a requirement for TP53 in this induced repair process. We find that in normal cells, the endogenous levels of TP53 are elevated at the G1/S-phase border, and that these levels are not further increased after irradiation with 5 Gy. In cells expressing the E6 oncoprotein of human papillomavirus, which inactivates TP53 function, there is a greatly accentuated induction of the VLRP that nearly masks the constitutive repair response. Incubation of cells in the presence of cycloheximide, which inhibits the induced repair, reveals the presence of the constitutive repair patches. All cells examined continue to replicate their DNA after exposure to ionizing radiation. In contrast, cells irradiated with UV radiation at the G1/S-phase border show an induction of TP53 protein and halt DNA synthesis, but do not induce the VLRP. Our results show that TP53 is not required for the constitutive or induced repair of DNA damage induced by ionizing radiation. In addition, these results suggest that TP53 may suppress the formation of VLRP and that the progression of cells through S phase after exposure to ionizing radiation signals the induced repair response.  相似文献   

19.
Abstract

On UV irradiation of Escherichia coli cells, DNA replication is transiently arrested to allow removal of DNA damage by DNA repair mechanisms. This is followed by a resumption of DNA replication, a major recovery function whose mechanism is poorly understood. During the post-UV irradiation period the SOS stress response is induced, giving rise to a multiplicity of phenomena, including UV mutagenesis. The prevailing model is that UV mutagenesis occurs by the filling in of single-stranded DNA gaps present opposite UV lesions in the irradiated chromosome. These gaps can be formed by the activity of DNA replication or repair on the damaged DNA. The gap filling involves polymerization through UV lesions (also termed bypass synthesis or error-prone repair) by DNA polymerase III. The primary source of mutations is the incorporation of incorrect nucleotides opposite lesions. UV mutagenesis is a genetically regulated process, and it requires the SOS-inducible proteins RecA, UmuD, and UmuC. It may represent a minor repair pathway or a genetic program to accelerate evolution of cells under environmental stress conditions.  相似文献   

20.
We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase α–primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg2+ and Mn2+. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号