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J. Biol. Chem., Vol. 262, Issue 5, 2066-2076, 02, 1987
SW Matson and JW George
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.
DNA helicase II of Escherichia coli. Characterization of the single- stranded DNA-dependent NTPase and helicase activities
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