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Nucleic Acids Research, 1992, Vol. 20, No. 21 5647-5653
© 1992

ENZYMOLOGY

Alteration by site-directed mutagenesis of the conserved lysine residue in the consensus ATP-binding sequence of the RecB protein of Escherichia coli

Susie Hsieh and Douglas A. Julin*

Department of Chemistry and Biochemistry, University of Maryland College Park, MD 20742, USA

* To whom correspondence should be addressed

Received August 12, 1992. Accepted October 9, 1992.

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.


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