DNA连接酶的结构与功能

在ＤＮＡ合成中 ,合成方向为 5′→ 3′ ,即一条链上是连续复制的 ,而另一条链的复制是不连续的 ,必须先合成岗崎片段 (真核细胞的岗崎片段为 10 0ｂｐ ,原核细胞的为 10 0 0ｂｐ)。ＤＮＡ连接酶的作用就是催化岗崎片段的连接以完成ＤＮＡ的合成。另外 ,ＤＮＡ连接酶在ＤＮＡ修复过程中也起重要作用 ,如在切除修复中 ,切除损伤的ＤＮＡ片段 ,以未受损伤的链作为模板合成一条新的ＤＮＡ链后 ,ＤＮＡ连接酶将新合成的ＤＮＡ链与原来的ＤＮＡ链之间的缺口封闭完成ＤＮＡ的修复。ＤＮＡ链未封闭的缺口对细胞具有潜在的危险性 ,所以 ,ＤＮＡ连接…     

线粒体DNA及其表达的研究进展

线粒体属于半自主性细胞器 ,含有环状ＤＮＡ ,能进行自我复制 (重组和修复机制也包括在内 )。但线粒体ＤＮＡ(ｍｔＤＮＡ)的复制仍受细胞核的控制 ,因为不仅构成线粒体的蛋白质几乎都受核基因编码、在细胞质中合成 ,而且与ｍｔＤＮＡ有关的特定蛋白质(如ＤＮＡ聚合酶、重组与修复所需的酶、ＲＮＡ聚合酶、ＲＮＡ加工酶 )也都是由核基因编码的。ｍｔＤＮＡ的复制、转录、翻译及蛋白质的输入有其特殊规律 ,阐明线粒体的分子遗传规律既有助于理解线粒体在凋亡或程序性细胞死亡中发挥的作用 ,因而可更深入地对发育生物学、癌症、老化及机体死亡…     

真核细胞DNA聚合酶

近年来,真核细胞ＤＮＡ体外复制模型的建立及蛋白纯化技术的提高,ＤＮＡ聚合酶基因的克隆,真核细胞ＤＮＡ聚合酶研究取得了很大进展。     

人源DNA聚合酶δ研究进展

在真核生物染色体DNA复制过程中主要涉及三种DNA聚合酶:α(Polα),δ(Polδ)和ε(Polε)。人源DNA聚合酶δ是p125,p68,p50,p12四个亚基构成的异源四聚体,属于DNA聚合酶B家族,具有5’-3’聚合酶催化活性和3’-5’核酸外切酶活性,是染色体DNA复制过程中最主要的复制酶,同时还参与多种形式的损伤修复,在保证基因组结构的完整性和遗传稳定性方面具有重要的意义。由于其重要的生物学功能,目前引起人们更多的关注和重视。对人源DNA聚合酶δ的分离纯化方法及涉及DNA复制和损伤修复过程中酶学功能等方面的最新研究进展进行综述。     

动物细胞DNA聚合酶的研究新进展

本文主要介绍动物细胞中四种依赖于DNA的DNA聚合酶(简称DNA聚合酶)的结构、功能及其在DNA复制和修复作用中的研究现状。     

真核细胞DNA聚合酶δ的结构与功能

DNA聚合酶δ(Polδ)在真核细胞的DNA复制过程中具有核心酶的作用,同时还参与DNA的修复。Polδ是一种由多个亚基组成的复合体,目前已从哺乳动物、裂殖酵母和芽殖酵母等多种真核生物细胞中分离出,并对它们的亚基组成进行了分析,但还未得到确切一致的结果。Polδ在DNA复制中的具体作用已基本了解,它参与催化整个前导链的复制以及一些或大部分滞后链的复制。此外,Polδ还参与DNA的修复,此酶的这一功能可减少DNA的变异,但目前对其作用机理还知之较少。在Polδ活性调控方面,主要研究了一些相关蛋白因子对Polδ活性的调控作用以及转录因子对催化亚基表达的调控作用。     

DNA连接酶Ⅲ在线粒体基因组完整性保持中的作用

真核DNA连接酶(DNA ligase)通过催化ATP依赖的双链DNA切口连接而在DNA复制、重组和修复过程中发挥了重要作用．DNA连接酶Ⅲ(Lig3)是一种独特性的连接酶,既可定位于细胞核,又可定位于线粒体．Lig3通过与DNA修复蛋白XRCC1作用而参与了碱基切除修复和其他单链断裂修复．但Lig3以XRCC1不依赖方式在线粒体DNA完整性保持方面发挥了更为重要的作用．这些研究为Lig3功能和DNA修复研究提供了新的视野．     

DNA Polymerases that Propagate the Eukaryotic DNA Replication Fork

Abstract

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase α -primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase α and recruits DNA polymerase δ and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase δ, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase ε normally replicates this strand, but under conditions of dysfunction, DNA polymerase δ may substitute.     

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.     

Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation

Chromosomal DNA replication requires one daughter strand-the lagging strand-to be synthesised as a series of discontinuous, RNA-primed Okazaki fragments, which must subsequently be matured into a single covalent DNA strand. Here, we describe the reconstitution of Okazaki fragment maturation in vitro using proteins derived from the archaeon Sulfolobus solfataricus. Six proteins are necessary and sufficient for coupled DNA synthesis, RNA primer removal and DNA ligation. PolB1, Fen1 and Lig1 provide the required catalytic activities, with coordination of their activities dependent upon the DNA sliding clamp, proliferating cell nuclear antigen (PCNA). S. solfataricus PCNA is a heterotrimer, with each subunit having a distinct specificity for binding PolB1, Fen1 or Lig1. Our data demonstrate that the most efficient coupling of activities occurs when a single PCNA ring organises PolB1, Fen1 and Lig1 into a complex.     

Mechanisms of single-stranded DNA-binding protein functioning in cellular DNA metabolism

This review deals with analysis of mechanisms involved in coordination of DNA replication and repair by SSB proteins; characteristics of eukaryotic, prokaryotic, and archaeal SSB proteins are considered, which made it possible to distinguish general mechanisms specific for functioning of proteins from organisms of different life domains. Mechanisms of SSB protein interactions with DNA during metabolism of the latter are studied; structural organization of the SSB protein complexes with DNA, as well as structural and functional peculiarities of different SSB proteins are analyzed.     

DNA polymerase proofreading: Multiple roles maintain genome stability

DNA polymerase proofreading is a spell-checking activity that enables DNA polymerases to remove newly made nucleotide incorporation errors from the primer terminus before further primer extension and also prevents translesion synthesis. DNA polymerase proofreading improves replication fidelity ∼ 100-fold, which is required by many organisms to prevent unacceptably high, life threatening mutation loads. DNA polymerase proofreading has been studied by geneticists and biochemists for > 35 years. A historical perspective and the basic features of DNA polymerase proofreading are described here, but the goal of this review is to present recent advances in the elucidation of the proofreading pathway and to describe roles of DNA polymerase proofreading beyond mismatch correction that are also important for maintaining genome stability.     

RNA:DNA hybrids from Okazaki fragments contribute to establish the Ku-mediated barrier to replication-fork degradation

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Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication

The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.Communicated by H. Ikeda     

Comprehensive Search for DNA Polymerase in the Hyperthermophilic Archaeon,Pyrococcus furiosus

DNA polymerase activities were scanned in a Pyrococcus furiosus cell extract to identify all of the DNA polymerases in this organism. Three main fractions containing DNA polymerizing activity were subjected to Western blot analyses, which revealed that the main activities in each fraction were derived from three previously identified DNA polymerases. PCNA (proliferating cell nuclear antigen), the sliding clamp of DNA polymerases, did not bind tightly to any of the three DNA polymerases. A primer usage preference was also shown for each purified DNA polymerase. Considering their biochemical properties, the roles of the three DNA polymerases during DNA replication in the cells are discussed.     

Essential functions of iron-requiring proteins in DNA replication,repair and cell cycle control

Eukaryotic cells contain numerous iron-requiring proteins such as iron-sulfur (Fe-S) cluster proteins, hemoproteins and ribonucleotide reductases (RNRs). These proteins utilize iron as a cofactor and perform key roles in DNA replication, DNA repair, metabolic catalysis, iron regulation and cell cycle progression. Disruption of iron homeostasis always impairs the functions of these ironrequiring proteins and is genetically associated with diseases characterized by DNA repair defects in mammals. Organisms have evolved multi-layered mechanisms to regulate iron balance to ensure genome stability and cell development. This review briefly provides current perspectives on iron homeostasis in yeast and mammals, and mainly summarizes the most recent understandings on iron-requiring protein functions involved in DNA stability maintenance and cell cycle control.     

大肠杆菌细胞DNA复制、修复和重组途径的衔接

以大肠杆菌为例围绕相关领域的研究动态进行分析和总结.DNA复制、损伤修复和重组过程的相互作用关系研究是当今生命科学研究的前沿和热点之一.越来越多的研究表明,在分子水平上,DNA复制、损伤修复和重组过程既彼此独立,又相互依存.上述途径可以通过许多关键蛋白质之间的相互作用加以协调和整合,并籍此使遗传物质DNA得到有效的维护和忠实的传递.需要指出的是,基于许多细胞内关键蛋白及其功能在生物界中普遍保守性的事实,相信来自大肠杆菌有关DNA复制、修复和重组之间的研究成果也会对相关真核生物的研究提供借鉴.