DNA helicase II of Escherichia coli. Characterization of the single-stranded DNA-dependent NTPase and helicase activities.
Escherichia coli helicase II has been purified to near homogeneity from cells harboring a multicopy plasmid containing the structural gene for helicase II, uvrD. In this paper a detailed description of the single-stranded DNA-dependent nucleoside 5'-triphosphatase and helicase reactions catalyzed by helicase II is presented. The results of this study suggest that nucleoside 5'-triphosphate hydrolysis provides the energy required for translocation of the enzyme along single-stranded DNA. Measurements of the rate of ATP hydrolysis using a variety of single-stranded DNAs of known structure and length suggest a processive translocation mechanism for helicase II. Single-stranded DNA coated with either Escherichia coli single-stranded DNA binding protein (SSB) or bacteriophage T4 gene 32 protein fails to support helicase II ATPase activity. Moreover, helicase II is apparently unable to displace a molecule of bound SSB protein from single-stranded DNA when it is encountered in the process of translocation along a single-stranded DNA effector. The helicase reaction has been characterized using an in vitro strand displacement helicase assay. The helicase reaction requires concomitant nucleoside 5'-triphosphatase hydrolysis that is satisfied by the hydrolysis of either rATP or dATP. As the length of duplex DNA present in the partial duplex helicase substrate is increased from 71 base pairs to 343 base pairs, the fraction of duplex DNA molecules that are unwound by helicase II decreases in the absence of any accessory proteins. However, the total number of base pairs of duplex DNA unwound depends primarily on the amount of enzyme added to the helicase reaction and not on the length of the duplex DNA present in the partial duplex DNA substrate. These data suggest the number of base pairs of duplex DNA unwound is directly proportional with the concentration of helicase II in the reaction mixture. In addition, the rate of the unwinding reaction is independent of the length of the duplex DNA available for unwinding. Helicase II has been shown to dissociate from single-stranded DNA molecules infrequently acting as an ATPase. However, the enzyme dissociates from partial duplex helicase substrates more frequently. This suggests a more distributive reaction mechanism on duplex DNA than was observed on single-stranded DNA substrates. The fraction of 343-base pair partial duplex DNA molecules unwound by helicase II can be increased by the addition of appropriate concentrations of E. coli SSB to the reaction. This suggests that helicase II and SSB may act in a concerted reaction to unwind duplex DNA.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D004265:DNA Helicases; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D013604:T-Phages / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases