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 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 chi-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 degrees 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 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.
Chen
H W
HW
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Ruan
B
B
Yu
M
M
Wang
J d
Jd
Julin
D A
DA
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D006639:Histidine; D006868:Hydrolysis; D011487:Protein Conformation
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases