Llosa M, Grandoso G, Hernando M A, de la Cruz F
Departamento de Biología Molecular, Facultad de Medicina, Santander, Spain.
J Mol Biol. 1996 Nov 22;264(1):56-67. doi: 10.1006/jmbi.1996.0623.
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.
TrwC是一种双功能酶，具有质粒R388接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。我们过量表达并纯化了该蛋白的不同片段，从而能够将松弛酶和DNA解旋酶活性定位到蛋白的不同区域。当使用包含nic位点的寡核苷酸时，由该蛋白N端275个氨基酸残基组成的肽能够催化DNA切割和链转移反应，不过在超螺旋双链DNA底物上产生切口需要更长的TrwC片段（348个氨基酸残基）。氨基酸残基192至966之间的蛋白片段包含ATP酶和DNA解旋酶活性，而由氨基酸残基346至966组成的肽则失去了这两种活性。二聚化区域位于C端495个氨基酸残基处。分别含有DNA链转移酶和DNA解旋酶活性的两种肽在R388接合过程中可以在功能上替代TrwC，尽管效率要低10,000倍。因此，高效的DNA转移需要蛋白共价结构的完整性。可以推测，共价连接通过增加一种组分（可能是DNA解旋酶）在其作用位点的有效浓度来提高接合效率。
