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

染色体DNA的复制是所有生命的基础，确保了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，现存的生命形式被分为三大领域：细菌、真核生物和古生物。在这三个领域，大量蛋白质的协同活动被用来实现严格调控、快速和准确的DNA复制。这些蛋白质组装成一个称为复制体的多蛋白质复合体。DNA聚合酶是负责实际复制DNA的酶，是关键的复制体组分。在细菌中，复制是最早被研究的领域，也是最被理解的领域，C家族DNA聚合酶(DNA聚合酶III)被用来复制DNA。然而，另外两个结构域，真核生物和古生菌缺乏POL III的同源基因，而它们的基因组主要编码B家族聚合酶。在真核生物中，一对B家族酶被用来复制这两条DNA链。所有古菌都含有至少一个B家族聚合酶，具有与DNA复制兼容的生化特性。通过与真核生物的类比，人们通常认为在古生菌中，普遍观察到的B-聚合酶负责复制。然而，在与俄亥俄州立大学的科学家(约翰·里夫教授和汤姆·桑坦格罗博士)的合作中，康诺利教授表明，从考古菌热球菌kodakarsis(Tkod)中移除单个家族B聚合酶是没有影响的。这种Tkod缺失菌株的生长速度与野生型相同，对DNA损伤剂具有同样的敏感性，并且在DNA复制过程中不会出现更多错误，这表明Pol-B对DNA复制不是关键。一种新的DNA聚合酶Pol-D在五个典型的古菌门(Eury-、thaum-、Kor-和Nanoterea)中的四个中被观察到，尽管在第五个门--crenchaceea中似乎缺少这种酶。POL-D还具有与DNA复制兼容的属性，并且与POL-B不同，它不能在Tkod中删除。这些观察结果提出了一种可能性，即在大多数古生物物种中，D家族聚合酶负责复制DNA。D家族聚合酶的特征很差，在亚单位结构方面表现出独特的特征(一种异二聚体，由一个大的聚合酶亚单位和一个小的校对核酸外切酶亚单位组成)，并且与其他(家族-B和-C)复制聚合酶几乎没有氨基酸相似性。因此，建议使用生化和遗传学相结合的方法来深入研究POL-D的性质和功能。我们将使用温和的方法来纯化该酶，以保护疑似存在于聚合酶中的金属辅因子(特别是铁-S簇，容易被氧破坏)。一整套体外实验将被用来确定这种酶如何复制DNA并对DNA损伤做出反应，它还有望确定一种高分辨率的结构。体内补充性实验，操纵遗传易受影响的甲烷球菌中的染色体Pol-D基因，将阐明这种酶在细胞中所起的作用。证明大多数古菌使用POL-D进行复制，因此生命的三个区域具有不同的复制聚合酶，这引发了关于DNA复制的进化和不同复制策略的优势的深刻问题。

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.