Mechanisims and Biological Significance of Recombination-dependent DNA Replication

(1)我们已经构建了在ATP结合结构域或在推定的锌指基序的半胱氨酸残基中含有点突变的PriA突变基因。(2)我们的初步结果表明，ATP结合和锌指基序的稳定DNA复制的重要作用。(3)PriA蛋白在体外与ColE 1型质粒起始处产生的R环结构结合。(4)N-末端300个氨基酸足以结合n '-pas，n'-pas是一种发夹，用作phiX 174型原小体的组装位点。(5)结果发现，支原体PriA同源基因含有锌指结构，但缺少部分ATP结合/解旋酶基序。(6)搜索酿酒酵母基因组的整个核苷酸序列没有发现与PriA具有令人信服的同源性的基因。这表明PriA的真核同源物的结构（如果有的话）可能与细菌PriA及其相关基因不同。(7)我们对依赖DNA A的A位点启动子进行了酶促鉴定，发现了一个新的可被RNA聚合酶全酶识别的单链DNA启动子，该启动子含有sigma^<70>。称为RPO位点，该序列作为下游基因的启动子，也作为DNA复制的引物RNA合成的位点。RPO位点是一个单链形式的启动子，在SSB存在下是一个活性启动子。

(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.