Escherichia coli Rep protein and helicase IV. Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro.
Rep protein and helicase IV, two DNA-dependent adenosine 5'-triphosphatases with helicase activity, have been purified from Escherichia coli and characterized. Both enzymes exhibit a distributive interaction with single-stranded DNA as DNA-dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E. coli single-stranded DNA binding protein (SSB). The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates. Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length. However, unwinding of a 119-bp or 343-bp duplex region was substantially reduced compared to unwinding of the 71-bp substrate. At each concentration of protein examined, the number of base pairs unwound was greatest using the 71-bp substrate, intermediate with the 119-bp substrate and lowest using the 343-bp substrate. The addition of E. coli SSB did not increase the fraction of the 343-nucleotide fragment unwound by Rep protein. However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold. In addition, ionic strength conditions which stabilize duplex DNA (i.e. addition of MgCl2 or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction. Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA. Taken together these data suggest that, in vitro, and in the absence of additional proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.
Yancey-Wrona
J E
JE
Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
Wood
E R
ER
George
J W
JW
Smith
K R
KR
Matson
S W
SW
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D066298:In Vitro Techniques; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D012265:Ribonucleotides / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D003854:Deoxyribonucleotides; D029968:Escherichia coli Proteins; D012265:Ribonucleotides; C506744:rep protein, E coli; D015636:Magnesium Chloride; D012965:Sodium Chloride; D000251:Adenosine Triphosphatases; D004265:DNA Helicases