Abstract
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.
Highlights
Escherichia coli cell extracts that significantly stimu- uitous in nature
TheRep helicase unwinding reac- stance, DNA helicase I is tion was stimulated byas much as aO-fold,upon addi- required for conjugalDNA transfer (Abdel-Monem et al, tion of the stimulatory protein, using either 7a1-base 1983),helicase I1 is involved in excision repair (Husain et al, pair or a 343-base pair partial duplex DNA molecule 1985;Caron et al, 1985)and methyl-directed mismatch repair as a substrate
Purification of the Rep Helicase Stimulatory Protein-Rep helicasestimulatory protein (RHSP) was purified from E. coli HF4704rep3 (Yarranton and Gefter, 1979a).The cells were grown, harvested, resuspended, and fractions I, 11, and 111 were prepared as described previously (Wood and Matson, 1987)
Summary
The abbreviations used are: NTP, nucleoside 5”triphosphate; CisA protein, cistron A protein; ssDNA, single-stranded DNA; bp, base pair; RHSP, Rep helicase stimulatory protein; SSB, E . This unexpected result, in view of the role ofRep protein in 4x174 DNA metabolism, has led to theidentification, purification, and partial characterization of a Rep protein stimulatory factor from E. coli cell extracts. The purified protein is small (Mr= 15,000) and apparently single-stranded DNA binding protein. The purified protein does not stimulate the unwinding reactions catalyzed by E. coli helicase I or the 75-kDa helicase
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