Hsieh S, Julin D A
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Nucleic Acids Res. 1992 Nov 11;20(21):5647-53. doi: 10.1093/nar/20.21.5647.
The RecB and RecD subunits of the RecBCD enzyme of Escherichia coli contain amino acid sequences similar to a consensus mononucleotide binding motif found in a large number of other enzymes. We have constructed by site-directed mutagenesis a lysine-to-glutamine mutation in this sequence in the RecB protein. The mutant enzyme (RecB-K29Q-CD) has essentially no nuclease or ATP hydrolysis activity on double-stranded DNA, showing the importance of RecB for unwinding double-stranded DNA. However, ATP hydrolysis stimulated by single-stranded DNA is reduced by only about 5-8-fold compared to the wild-type, nuclease activity on single-stranded DNA is reduced by less than 2-fold, and the nuclease activity of the RecB-K29Q-CD enzyme requires ATP. The effects of the RecB mutation suggest that the RecD protein hydrolyzes ATP and can stimulate the RecBCD enzyme nuclease activity on single-stranded DNA.
大肠杆菌RecBCD酶的RecB和RecD亚基含有与大量其他酶中发现的共有单核苷酸结合基序相似的氨基酸序列。我们通过定点诱变在RecB蛋白的该序列中构建了赖氨酸到谷氨酰胺的突变。突变酶（RecB-K29Q-CD）对双链DNA基本上没有核酸酶或ATP水解活性，这表明RecB对于解开双链DNA很重要。然而，与野生型相比，单链DNA刺激的ATP水解仅降低了约5-8倍，对单链DNA的核酸酶活性降低不到2倍，并且RecB-K29Q-CD酶的核酸酶活性需要ATP。RecB突变的影响表明，RecD蛋白水解ATP并可以刺激RecBCD酶对单链DNA的核酸酶活性。
