The RecD Subunit of the RecBCD Enzyme from Escherichia coli Is a Single-stranded DNA-dependent ATPase*
- From the ‡ Department of Chemistry and
- § Program in Molecular and Cell Biology, University of Maryland, College Park, Maryland 20742
- ↵∥ To whom correspondence should be addressed. Tel.: 301-405-1821; Fax: 301-314-9121; E-mail: dj13{at}umail.umd.edu.
Abstract
We have expressed the RecD subunit of the RecBCD enzyme from Escherichia coli as a fusion protein with a 31-amino acid NH2-terminal extension including 6 consecutive histidine residues (HisRecD). The overexpressed fusion protein can be purified in urea-denatured form by metal chelate affinity chromatography. The mixture of renatured HisRecD protein and the RecB and RecC proteins has a high level of ATP-dependent nuclease activity with either single- or double-stranded DNA, enhanced DNA unwinding activity, enhanced ATP hydrolysis activity in the presence of a small DNA oligomer cosubstrate, and χ-cutting activity. These are all characteristics of the RecBCD holoenzyme. The HisRecD protein by itself hydrolyzes ATP in the presence of high concentrations of single-stranded DNA (polydeoxythymidine). The activity is unstable at 37°C, but is measurable at room temperature (about 23°C). The HisRecD has very little ATPase activity in the presence of a much shorter single-stranded DNA (oligodeoxy(thymidine)12). HisRecD hydrolyzes ATP more efficiently than GTP and UTP, and has very little activity with CTP. We also purified a fusion protein containing a Lys to Gln mutation in the putative ATP-binding site of RecD. This mutant protein has no ATPase activity, indicating that the observed ATP hydrolysis activity is intrinsic to the RecD protein itself.
Footnotes
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↵* This research was supported by Grant GM39777 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received November 11, 1996.
- Revision received February 3, 1997.
- © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
