THE RECD SUBUNIT OF THE RECBCD ENZYME FROM ESCHERICHIA-COLI IS A SINGLE-STRANDED DNA-DEPENDENT ATPASE

We have expressed the RecD subunit of the RecBCD enzyme from Escherich ia coli as a fusion protein with a 31-amino acid NH2-terminal extensio n including 6 consecutive histidine residues (HisRecD). The overexpres sed fusion protein can be purified in urea-denatured form by metal che late affinity chromatography. The mixture of renatured HisRecD protein and the RecB and RecC proteins has a high level of ATP-dependent nucl ease 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 chi-cutting activity. These a re all characteristics of the RecBCD holoenzyme. The HisRecD protein b y itself hydrolyzes ATP in the presence of high concentrations of sing le-stranded DNA (polydeoxythymidine). The activity is unstable at 37 d egrees C, but is measurable at room temperature (about 23 degrees C). The HisRecD has very little ATPase activity in the presence of a much shorter single-stranded DNA (oligodeoxy(thymidine)(12)). HisRecD hydro lyzes ATP more efficiently than GTP and UTP, and has very little activ ity with GTP. We also purified a fusion protein containing a Lys to Gl n mutation in the putative ATP-binding site of RecD. This mutant prote in has no ATPase activity, indicating that the observed ATP hydrolysis activity is intrinsic to the RecD protein itself.