COMPUTER-ASSISTED DISSECTION OF ROLLING CIRCLE DNA REPLICATION

起始蛋白氨基酸序列的比较分析 和复制起点的核苷酸序列 进行环状DNA复制（RCR）以预测 未鉴定的功能位点，包括共价连接的氨基酸残基 并评估复制子之间的关系 利用RCR。 RCR是DNA复制的基本机制之一 被许多小的单链和双链DNA利用 真细菌和古细菌的复制子，并由至少两组 小型真核病毒、细小病毒和双生病毒。详细 比较RCR起始蛋白的氨基酸序列， 由序列组成的基序的鉴定 HisHydHisHydHydHydHydr（Hyd-bulky疏水性残基），并且是保守的 在两大类蛋白质中，其中一类涉及RCR本身， (Rep蛋白质），另一个在动员（配偶转移）， 质粒DNA（Mob蛋白）。基于与金属酶的类比， 假设在这个基序中的两个保守的His残基可能是 参与Rep活性所需的金属离子配位 和Mob蛋白质。Rep蛋白含有两个额外的保守基序， 其中一个位于上游，另一个位于下游， 他的”母题。C-末端基序包括形成酪氨酸残基的Tyr残基。 与切口DNA的共价连接。Mob蛋白的特征在于： 保守基序的相反方向，与（假定的） DNA连接Tyr位于它们的N-末端附近。代表和暴民 蛋白质类进一步分成几个不同的家族。虽然 不可能找到一个独特的图案或图案 整个Rep或Mob类，为大型子集导出独特模式 每一种蛋白质。这些观察结果使得预测 参与DNA切口的氨基酸残基，这是必需的， 在Rep中，RCR的起始或-链ssDNA的接合转移， 由许多大小高度不同的复制子编码的Mob蛋白， 结构和起源。据证实，重组发挥了重要作用。 编码相关Rep或Mob的基因传播的主要部分 利用RCR的复制子中的蛋白质。外界猜测 真核小ssDNA复制子编码具有保守RCR的蛋白质 基序和通过RCR相关机制复制，即双生病毒和 细小病毒，可能是从真细菌复制子进化而来的。 该项目的 重要性在于预测在许多细胞中的功能位点。 广泛研究的蛋白质和明显的进化证据 许多不同的真细菌、古细菌和 真核复制子实验旨在测试的预测， 细小病毒复制起始蛋白中的功能位点位于 进展D.塔特萨尔的实验室

Comparative analysis of the amino acid sequences of the initiator proteins and of the nucleotide sequences of the replication origins for rolling circle DNA replication (RCR) was performed in order to predict unidentified functional sites including amino acid residue covalently linking to DNA and to assess the relationships between replicons exploiting RCR. RCR is one of the basic mechanisms of DNA replication exploited by numerous small single-stranded (ss)and double-stranded DNA replicons from eubacteria and archaebacteria, and by at least two groups of small eukaryotic viruses, parvorisuses and geminiviruses. Detailed comparisons of the amino acid sequences of RCR initiator proteins led to the identification of a motif that consists of the sequence HisHydrHisHydrHydrHydr (Hydr-bulky hydrophobic residue) and is conserved in two vast classes of proteins, one of which is involved in RCR proper (Rep proteins), and the other in mobilization (conjugal transfer)of plasmid DNA (Mob proteins). Based on analogies with metalloenzymes, it is hypothesized that the two conserved His residues in this motif may be involved in metal ion coordination required for the activity of the Rep and Mob proteins. Rep proteins contained two additional conserved motifs, one of which was located upstream, and the other downstream from the "two His" motif. The C-terminal motif encompassed the Tyr residue(s) forming the covalent link with nicked DNA. Mob proteins were characterized by the opposite orientation of the conserved motifs, with the (putative) DNA-linking Tyr being located near their N-termini. Both Rep and Mob protein classes further split into several distinct families. Although it was not possible to find a motif or pattern that would be unique for the entire Rep or Mob class, unique patterns were derived for large subsets of the proteins of each class. These observations allowed the prediction of the amino acid residues involved in DNA nicking, which is required for the initiation of RCR or conjugal transfer of -stranded ssDNA, in Rep and Mob proteins encoded by a number of replicons of highly diverse size, structure and origin. It is conjectured that recombination has played a major part in the dissemination of genes encoding related Rep or Mob proteins among the replicons exploiting RCR. It is speculated that the eukaryotic small ssDNA replicons encoding proteins with the conserved RCR motifs & replicating via RCR-related mechanisms, i.e. geminiviruses & parvoviruses, may have evolved from eubacterial replicons. The project's significance lies in prediction of the functional sites in a number of widely studied proteins and the demonstration of apparent evolutionary links between a number of diverse eubacterial, archaebacterial, & eukaryotic replicons. Experiments designed to test the predictions of the functional sites in the parvovirus replication initiation proteins are in progress in D. Tattersall's lab.