Zhang G, Deng E, Baugh L R, Hamilton C M, Maples V F, Kushner S R
Department of Genetics, University of Georgia, Athens 30602, USA.
J Bacteriol. 1997 Dec;179(23):7544-50. doi: 10.1128/jb.179.23.7544-7550.1997.
There are seven conserved motifs (IA, IB, and II to VI) in DNA helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.
大肠杆菌DNA解旋酶II中有七个保守基序（IA、IB以及II至VI），在参与DNA代谢的一大类蛋白质中具有高度同源性。为了探究基序II至VI的功能重要性，我们采用定点诱变，将每个基序中的带电荷氨基酸残基替换为丙氨酸。携带这些突变等位基因的细胞对紫外线和甲磺酸甲酯表现出更高的敏感性，自发诱变率增加，同源重组水平升高，表明切除修复和错配修复途径均存在缺陷。此外，我们还将基序VI中高度保守的酪氨酸（600）替换为苯丙氨酸（uvrD309，Y600F）。该突变体对紫外线的敏感性适度增加，但自发突变率降低，这表明DNA解旋酶II在两种DNA修复途径中可能具有不同功能。此外，结构域IV中的一个突变（uvrD307，R284A）显著降低了一些大肠杆菌K-12菌株在30摄氏度时的活力，但在37摄氏度时并未降低。我们对这些观察结果的意义进行了讨论。
