Mechanisims and Biological Significance of Recombination-dependent DNA Replication

(1)我们构建了包含ATP结合域或锌指基序半胱氨酸残基点突变的PriA突变基因。(2)初步结果表明，atp结合基序和锌指基序在DNA稳定复制中都起着重要作用。(3) PriA蛋白在体外与cole1型质粒起源处产生的R-loop结构结合。(4) n端300个氨基酸足以与n'-pas结合，n'-pas是一个发夹，是phix174型引体的组装位点。(5)通过数据库检索，鉴定出6个与大肠杆菌PriA结构相关的细菌基因。有趣的是，假定的PriA支原体同源物缺乏部分ATP结合/解旋酶基序，尽管它包含保守的锌指基序。(6)对酿酒酵母基因组的全核苷酸序列进行搜索，没有发现与PriA具有令人信服的同源性的基因。这表明真核生物的PriA同源物（如果有的话）的结构可能与细菌的PriA及其相关基因不同。(7)我们对装配在A位点的dna依赖性原体进行了酶学表征。我们发现了一种新的单链DNA启动子，可以被RNA聚合酶全酶识别，该全酶含有sigma^<70>。该序列被称为RPO位点，是下游基因的启动子，也是DNA复制的引物RNA合成位点。RPO位点是单链形式的活性启动子，在SSB存在的情况下。我们已经阐明了RNA聚合酶对RPO位点独特二级结构的识别模式。

(1) We have constructed PriA mutant genes containng point mutations in the ATP binding domain or in the cysteine residues of the putative Zinc-finger motif.(2) Our preliminary results indicated essential roles of both ATP-binding and Zinc-finger motifs for stable DNA replication.(3) PriA protein binds to the R-loop structure generated at the origin of ColE1-type plasmid in vitro.(4) The N-terminal 300 amino acids are sufficient for binding to n'-pas, a hairpin which serves as an assembly site for the phiX174-type primosome.(5) Serach of data base lead to identificatin of six bacterial genes structurally related to E.coli PriA.Interestingly, the putative Mycoplasma PriA homologue lacks a part of the ATP binding/helicase motif, although it contains the conserved zinc-finger motif.(6) Search of the entire nucleotide sequence of the Saccharomyces cerevisiae genome did not result in identification of a gene with convincing homology to PriA.This indicates that the structure of an eukaryotic homologue of PriA,if any, may have diverged from bacterial PriA and its related genes.(7) We have enzymatically characterized DnaA-dependent primosome assembled at the A site.We have discovered a novel single-stranded DNA promoter recognized by RNA polymerase holoenzyme containing sigma^<70>. Termed RPO site, this sequence serves as a promoter for the genes downstream and also as the site for primer RNA synthesis for DNA replication. RPO site is an active promoter in the form of single-strand and in the presence of SSB.We have elucidated a mode of recognition of a unique secondary structure of RPO site by RNA polymerase.