Precise Determination, Cross-Recognition, and Functional Analysis of the Double-Strand Origins of the Rolling-Circle Replication Plasmids in Haloarchaea
Precise Determination, Cross-Recognition, and Functional Analysis of the Double-Strand Origins of the Rolling-Circle Replication Plasmids in Haloarchaea
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DOI：
10.1128/jb.00596-08
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发表时间：
2008-06
影响因子：
3.2
通讯作者：
Ligang Zhou;Meixian Zhou;Chaomin Sun;Jing Han;Qiuhe Lu;Jian Zhou;H. Xiang
中科院分区：
文献类型：
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作者：
Ligang Zhou;Meixian Zhou;Chaomin Sun;Jing Han;Qiuhe Lu;Jian Zhou;H. Xiang
摘要采用电子显微镜和DSO定位技术，确定了盐古菌Natrinema sp. CX2021中1668 bp滚圈复制（RCR）质粒pZMX201双链起源（DSO）的精确缺口位点。在该质粒中，DSO缺口发生在位于不完美发夹结构茎区的七核苷酸序列（TCTC/GGC）的C404和G405残基之间。该缺口位点序列在包括pNB101在内的盐古菌RCR质粒中是保守的，这表明DSO缺口位点可能在该质粒家族的所有成员中是相同的。有趣的是，在杂交质粒系统中，pZMX201和pNB101的dso在RCR启动和终止中被交叉识别。pZMX201 （DSOZ）的DSO在该杂交质粒体系中的突变分析表明：(1)保守的TCTCGGC序列中间的核苷酸在起始和终止过程中发挥更重要的作用；（ii）发夹结构的左半部分用于起始，但不用于终止；（iii）包含TCTCGGC和下游序列的36bp序列对于终止是必要和足够的。综上所述，这些盐古菌质粒具有不同于单链DNA噬菌体和细菌RCR质粒的新特征，可作为研究RCR复制子进化的良好模型。
ABSTRACT The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSOZ) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.