共查询到20条相似文献,搜索用时 11 毫秒
1.
Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations. 相似文献
2.
Model for DNA replication by Kornberg's DNA polymerase 总被引:3,自引:0,他引:3
A R Morgan 《Nature》1970,227(5265):1310-1313
3.
A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand. 相似文献
4.
5.
Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint 总被引:51,自引:0,他引:51
The checkpoint kinase proteins Mec1 and Rad53 are required in the budding yeast, Saccharomyces cerevisiae, to maintain cell viability in the presence of drugs causing damage to DNA or arrest of DNA replication forks. It is thought that they act by inhibiting cell cycle progression, allowing time for DNA repair to take place. Mec1 and Rad53 also slow S phase progression in response to DNA alkylation, although the mechanism for this and its relative importance in protecting cells from DNA damage have not been determined. Here we show that the DNA-alkylating agent methyl methanesulphonate (MMS) profoundly reduces the rate of DNA replication fork progression; however, this moderation does not require Rad53 or Mec1. The accelerated S phase in checkpoint mutants, therefore, is primarily a consequence of inappropriate initiation events. Wild-type cells ultimately complete DNA replication in the presence of MMS. In contrast, replication forks in checkpoint mutants collapse irreversibly at high rates. Moreover, the cytotoxicity of MMS in checkpoint mutants occurs specifically when cells are allowed to enter S phase with DNA damage. Thus, preventing damage-induced DNA replication fork catastrophe seems to be a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation. 相似文献
6.
7.
DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA-ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA-ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed 'intersegmental contact sampling', in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins. 相似文献
8.
9.
耐热DNA聚合酶介导的DNA酶促自发合成 总被引:1,自引:0,他引:1
构建了无模板引物的“类PCR体系”。该体系在适当温度保温一段时间之后,能检测到产物出现,利用热变性分析等手段,初步研究了产物的性质,发现这些产物是随机合成的具有一些特殊构型的DNA。本文报道三种构型,两类碱基组成比例,并通过改变反应条件观察对酶促自发合成的影响,探讨耐热DNA聚合酶的非特异聚合活性以及介导的酶促自发合成的机理和意义。 相似文献
10.
彗星电泳检测细胞DNA损伤应用新进展 总被引:3,自引:0,他引:3
彗星电泳是近年发展起来的一种测定单个细胞DNA损伤的方法 ,因其操作简单、快速及灵敏等特点 ,应用范围十分广泛 .着重介绍了这一技术的发展、图形分析及应用进展 相似文献
11.
In order to investigate ATM in mediating DNA damage signal to p53 in the cellular response to IR, kinase activities of ATM
and c-AbI immunoprecipitates and its activation by IR and damaged DNA have been analyzed. Results demonstrate that deficient
ATM caused failure to activate phosphorylation of many proteins in response to radiation. ATM coimmunoprecipitates with c-AbI
and can catalyze phosphorylation of many proteins including p53 in response to radiation. Kinase activity of ATM / c-AbI immunoprecipitate
stimulated by damaged DNAin vitro phosphorylation demonstrates that ATM can detect damaged DNA and initiate DNA damage signals. ATM can be phosphorylatedin vitro and inhibited by wortmannin, a specific inhibitor of PI3K family. These results confirm that ATM acts in DNA damage detection
and signal transduction. 相似文献
12.
P C Hanawalt 《Nature》1967,214(5085):269-270
13.
Escherichia coli contains three DNA polymerases that differ in their size, ability to interact with accessory proteins and biological function. Monomeric DNA polymerase I (Pol I) has a relative molecular mass (Mr) of 103,000 (103K) and is involved primarily in the repair of damaged DNA and the processing of Okazaki fragments; polymerase II is of Mr 120K, and polymerase III has a Mr of 140K, is responsible for the replication of the DNA chromosome and is just one of several proteins that are required for replication. DNA polymerases from bacteriophage as well as those of eukaryotic viral and cellular origin also differ with respect to their size and the number of associated proteins that are required for them to function in replication. However, the template-directed copying of DNA is identical in all cases. The crystal structure of the large proteolytic fragment of Pol I shows that it consists of two domains, the larger of which contains a deep crevice whose dimensions are such that it can bind duplex DNA. The T7 polymerase consists of two subunits, the 80K gene 5 protein and the host-encoded 12K thioredoxin of E. coli. We show here that there is an amino acid sequence homology between at least eight polypeptide segments that form the large cleft in the Klenow fragment and polypeptides in T7 DNA polymerase gene 5 protein, suggesting that this domain evolved from a common precursor. The parts of the Pol I and T7 DNA polymerase molecules that bind the DNA substrate appear to share common structural features, and these features may be shared by all of these varied DNA polymerases. 相似文献
14.
Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin 总被引:96,自引:0,他引:96
Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented. 相似文献
15.
PCNA connects DNA replication to epigenetic inheritance in yeast 总被引:29,自引:0,他引:29
16.
Breaks in DNA stimulate transcription by core RNA polymerase 总被引:8,自引:0,他引:8
17.
18.
19.
20.
用聚合酶链式反应(PCR)法检测患者尿液中人巨细胞病毒(HCMV)DNA.结果表明,自行设计合成的引物位于HCMV基因组早期蛋白基因区,经PCR仪扩增一段长430bp的特异序列片段,对正常人基因组DNA或其它疱疹病毒DNA无交叉反应.此法可检测出少至10-16g(0.1fg)的病毒DNA.通过对35份尿液标本的检测,比较PCR法和组织培养法的检测结果完全一致 相似文献