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A rolling circle replication initiator protein with a nucleotidyl-transferase activity encoded by the plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi.

机译:由来自超嗜热古生毕赤酵母的质粒pGT5编码的具有核苷酸基转移酶活性的滚环复制起始蛋白。

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The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicate by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking-closing activity in vitro on single-stranded oligonucleotides containing the putative double-stranded replication origin sequence of pGT5. Gel shift analyses on single-stranded oligonucleotides indicate that Rep75 recognizes the single-stranded DNA region upstream of the nicking site via non-covalent interaction and remains covalently linked to the 5'-phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 contains a dATP (and ATP) terminal transferase activity at the 3'-OH extremity of the nicking site, which had not been reported previously for proteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model for the study of rolling circle replication.
机译:来自超嗜热古生火球菌的质粒pGT5与通过滚环机理复制的pC194家族的质粒具有相似性。这些质粒编码复制起始蛋白,该复制起始蛋白通过刻蚀两条DNA链之一来激活复制起点。已在大肠杆菌中克隆了编码pGT5假定的Rep蛋白的基因(Rep75),并在大肠杆菌中过表达,并且已将重组蛋白纯化至同质。 Rep75对含有推定的pGT5双链复制起点序列的单链寡核苷酸在体外表现出高度嗜热的切口关闭活性。对单链寡核苷酸的凝胶位移分析表明,Rep75通过非共价相互作用识别切刻位点上游的单链DNA区域,并且在切刻后仍与下游片段的5'-磷酸共价连接。除了这些预期的活性外,Rep75在切刻位点的3'-OH末端还具有dATP(和ATP)末端转移酶活性,以前尚未对此类型的蛋白质进行过报道。 Rep75是首个以古细菌为特征的复制起始蛋白,为滚动环复制的研究提供了有吸引力的新模型。

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