Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein.
A polypeptide (Mr = 15,000) has been purified from Escherichia coli cell extracts that significantly stimulates the duplex DNA unwinding reaction catalyzed by E. coli Rep protein. The Rep helicase unwinding reaction was stimulated by as much as 20-fold, upon addition of the stimulatory protein, using either a 71-base pair or a 343-base pair partial duplex DNA molecule as a substrate. The purified Rep helicase stimulatory protein (RHSP) had no intrinsic helicase activity or ATP hydrolysis activity and did not stimulate the single-stranded DNA-dependent ATP hydrolysis reaction catalyzed by Rep protein. It is likely that RHSP stimulates the Rep helicase unwinding reaction by stoichiometric binding to single-stranded DNA. However, a specific interaction between Rep protein and RHSP cannot be ruled out, since RHSP did not stimulate the duplex DNA unwinding reactions catalyzed by E. coli helicase I or the recently discovered 75-kDa helicase. RHSP did stimulate the duplex DNA unwinding reaction catalyzed by E. coli helicase II. The identification and subsequent purification of RHSP from cell extracts demonstrates the feasibility of using direct helicase assays to purify stimulatory proteins.
Smith
K R
KR
Department of Biology, University of North Carolina, Chapel Hill 27599.
Yancey
J E
JE
Matson
S W
SW
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000502:physiology; D004265:DNA Helicases / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004338:Drug Combinations; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008970:Molecular Weight
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004338:Drug Combinations; D029968:Escherichia coli Proteins; C506744:rep protein, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases