Functional domains in protein TrwC of plasmid R388: Dissected DNA strand transferase and DNA helicase activities reconstitute protein function
Functional domains in protein TrwC of plasmid R388: Dissected DNA strand transferase and DNA helicase activities reconstitute protein function
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DOI：
10.1006/jmbi.1996.0623
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发表时间：
1996-11-22
影响因子：
5.6
通讯作者：
delaCruz, F
中科院分区：
文献类型：
--
作者：
Llosa, M;Grandoso, G;delaCruz, F
TrwC是一种双功能酶，其显示质粒R388缀合所必需的两种生化活性：oriT特异性DNA链转移酶和DNA解旋酶活性。我们过量生产和纯化了蛋白质的不同片段，使我们能够将松弛酶和DNA解旋酶活性映射到蛋白质的不同区域。包含N-末端275个氨基酸残基的蛋白质的肽能够催化DNA切割和链转移反应时，使用寡核苷酸涵盖的nic网站，虽然需要一个较长的片段的TrwC（348个氨基酸残基），以产生切口上的超螺旋双链DNA基板。氨基酸残基192和966之间的蛋白质片段含有ATP酶和DNA解旋酶活性，而由氨基酸残基346至966组成的肽失去了这两种活性。二聚区位于495个C-末端氨基酸残基。两种分别含有DNA链转移酶和DNA解旋酶活性的肽可以在R388缀合中功能性地替代TrwC，尽管效率低10，000倍。因此，蛋白质共价结构的完整性是有效DNA转移所必需的。可以假设共价键通过增加一种组分（推测为DNA解旋酶）在其作用位点的有效浓度来增加缀合效率。(C)1996年学术出版社
TrwC is a bifunctional enzyme that displays two biochemical activities essential for plasmid R388 conjugation: oriT-specific DNA strand-transferase and DNA helicase activities. We overproduced and purified different segments of the protein allowing us to map the relaxase and DNA helicase activities to separate regions of the protein. A peptide comprising the N-terminal 275 amino acid residues of the protein was able to catalyze DNA cleavage and strand-transfer reactions when using oligonucleotides encompassing the nic site, although a longer fragment of TrwC (348 amino acid residues) was required to produce the nick on a supercoiled double-stranded DNA substrate. The segment of the protein between amino acid residues 192 and 966 contained the ATPase and DNA helicase activities, while a peptide consisting of amino acid residues 346 to 966 lost both activities. The dimerization region lay in the 495 C-terminal amino acid residues. Two peptides containing the DNA strand-transferase and DNA helicase activities, respectively, could functionally substitute for TrwC in R388 conjugation although at a 10,000-fold lower efficiency. Thus, integrity of the covalent structure of the protein was required for efficient DNA transfer. It can be assumed that the covalent linkage increases the efficiency of conjugation by increasing the effective concentration of one component (presumably the DNA helicase) at its site of action. (C) 1996 Academic Press Limited