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Biochemical and genetic characterisation of DNA polymerase D, a novel archaeal replicative polymerase

DNA 聚合酶 D（一种新型古菌复制聚合酶）的生化和遗传特征

基本信息

批准号：
BB/K005359/1
负责人：
Bernard Connolly
金额：
$ 46.34万
依托单位：
Newcastle University
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2013
资助国家：
英国
起止时间：
2013 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FK005359%2F1
关键词：
Biochemical genetic characterisation DNA polymerase

项目摘要

The replication of chromosomal DNA is fundamental to all life, ensuring the accurate transmission of genetic information from parent to progeny. All living organisms evolved from a single common ancestor and extant life forms are classified into three large domains, bacteria, eukarya and archaea. In all three domains the co-ordinated activity of a large number of proteins, assembled as a multi-protein complex called the replisome, is used to bring about tightly regulated, rapid and accurate copying of DNA. DNA polymerases, the enzymes responsible for actual copying of DNA, are a key replisome component. In bacteria, the domain in which replication was first studied and still the best understood, a C-family DNA polymerase (DNA polymerase III) is used to copy DNA. However, the two other domains, eukarya and archaea lack homologues of Pol III, rather their genomes encode primarily family-B polymerases. In eukaryotes a pair of family-B enzymes are used to copy the two DNA strands. All archaea contain at least one family-B polymerase, with biochemical properties compatible with DNA replication. By analogy with eukaryotes, it has commonly been assumed that in archaea, the universally observed B-polymerase is responsible for replication. However, in a collaboration with scientists (Prof. John Reeve and Dr. Tom Santangelo) at Ohio State University, Professor Connolly has shown that removal of the single family-B polymerase from the archaeon Thermococcus kodakarensis (Tkod) is without influence. This Tkod deletion strain grows at the same rate as the wild type, has the same sensitivity to DNA damaging reagents and does not make more errors during DNA replication, suggesting Pol-B is not critical for DNA replication. A novel DNA polymerase, Pol-D, has been observed in four of the five characterised archaeal phyla (eury-, thaum-, kor- and nanoarchaea), although the enzyme appears to be missing from the fifth phylum, the crenarchaea. Pol-D also has properties compatible with DNA replication and, unlike, Pol-B, cannot be deleted in Tkod. These observations raise the possibility that, in most archaeal species, the family-D polymerase is responsible for copying DNA. The family-D polymerases are poorly characterised and appear unique in terms of sub-unit structure (a heterodimer consisting of a large, polymerase, sub-unit and a small, proof reading exonuclease, sub-unit) and have little amino acid similarity with other (family-B and -C) replicative polymerases. It is, therefore, proposed to thoroughly investigate the properties and functions of Pol-D using a combination of biochemical and genetic methods. We will purify the enzyme using gentle approaches that should preserve the metallo-cofactors (notably an Fe-S cluster, susceptible to destruction by oxygen) suspected to be present in the polymerase. A full set of in vitro experiments will be used to determine how the enzyme copies DNA and responds to DNA damage and it is also hoped to determine a high resolution structure. Complementary in vivo experiments, manipulating the chromosomal Pol-D genes in the genetically tractable archaeon Methanococcus maripaludis, will elucidate the role the enzyme plays in the cell. Demonstrating that most archaea use Pol-D for replication, and so that the three domains of life have a different replicative polymerase, raises profound questions about the evolution of DNA replication and the advantages of different replicative strategies.

染色体DNA的复制是所有生命的基础，确保遗传信息从父母到后代的准确传递。所有的生物都是从一个共同的祖先进化而来的，现存的生命形式分为三大类：细菌、真核生物和古生菌。在这三个结构域中，大量蛋白质的协同活动被组装成称为复制体的多蛋白质复合物，用于实现严格调控、快速和准确的DNA复制。DNA聚合酶是负责DNA实际复制的酶，是复制体的关键组分。在细菌中，复制首先被研究并且仍然是最好理解的领域，C家族DNA聚合酶（DNA聚合酶III）用于复制DNA。然而，其他两个域，真核生物和古细菌缺乏同源物的Pol III，而他们的基因组编码主要是家庭B聚合酶。在真核生物中，一对B家族酶用于复制两条DNA链。所有古细菌都含有至少一种B族聚合酶，其生化特性与DNA复制相容。通过与真核生物类比，人们通常认为在古细菌中，普遍观察到的B-聚合酶负责复制。然而，在与俄亥俄州州立大学的科学家（John Reeve教授和Tom Santangelo博士）的合作中，Connolly教授已经表明，从古细菌Thermococcus kodakarensis（Tkod）中去除单一家族B聚合酶没有影响。这种Tkod缺失菌株以与野生型相同的速率生长，对DNA损伤试剂具有相同的敏感性，并且在DNA复制期间不会产生更多的错误，这表明Pol-B对DNA复制并不重要。一种新的DNA聚合酶，Pol-D，已被观察到的五个特征的古菌门（eury-，thaum-，kor-和nanoarchaea）中的四个，虽然酶似乎是失踪的第五门，crenarchaea。Pol-D也具有与DNA复制相容的特性，并且与Pol-B不同，Pol-D不能在Tkod中被删除。这些观察结果提出了一种可能性，即在大多数古细菌物种中，D家族聚合酶负责复制DNA。D家族聚合酶的特征很差，并且在亚单位结构（由大的聚合酶亚单位和小的证明阅读核酸外切酶亚单位组成的异源二聚体）方面看起来是独特的，并且与其他（B和C家族）复制型聚合酶几乎没有氨基酸相似性。因此，建议使用生物化学和遗传学方法的组合来彻底研究Pol-D的性质和功能。我们将使用温和的方法纯化酶，该方法应保留怀疑存在于聚合酶中的金属辅因子（特别是Fe-S簇，容易被氧破坏）。一整套体外实验将用于确定酶如何复制DNA并对DNA损伤作出反应，还希望确定高分辨率结构。互补的体内实验，操纵染色体Pol-D基因在遗传上易处理的古细菌甲烷球菌maripaludis，将阐明酶在细胞中发挥的作用。证明大多数古生菌使用Pol-D进行复制，因此生命的三个领域具有不同的复制聚合酶，提出了关于DNA复制进化和不同复制策略优势的深刻问题。