Abstract
Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3' to 5' direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.
Highlights
Any DNA unwinding that is observed in such a single turnover experiment must be catalyzed by the form of the enzyme that was prebound to the DNA because the trap prevents additional enzyme from binding to the DNA
Multiple turnover DNA unwinding experiments have been the most common method used to examine helicase activity. Such multiple turnover kinetics experiments cannot provide an unambiguous answer to this question because enzyme dissociation and reassociation can occur during the reaction, and the oligomeric state of the enzyme involved in the unwinding process is not known with certainty and even can change during the multiple turnover reaction. Using such single turnover DNA unwinding experiments, it has been shown that the SF1 enzymes E. coli Rep [22, 23] and E. coli UvrD (24 –26) require oligomerization to function as helicases in vitro, even though monomers of both of these enzymes are able to translocate with 3Ј to 5Ј directionality along ssDNA rapidly and processively [7, 8, 23]
PcrA Monomer Translocation along ssDNA—It was shown previously that monomers of B. stearothermophilus PcrA are able to translocate with biased 3Ј to 5Ј directionality along ssDNA [5, 6]
Summary
(30, 31), the phage ⌽x174 gene A protein increases the helicase processivity of E. coli Rep [32,33,34], and the RepD protein is a processivity factor for PcrA [35, 36]. The third is that only PcrA monomers are observed in the absence of DNA when examined by gel filtration at high salt concentrations (200 mM NaCl) [45] Based on these results it has been assumed that a PcrA monomer must be responsible for helicase activity in vitro. In this report we examine the ssDNA translocation and helicase activities of the B. stearothermophilus PcrA monomer using single turnover kinetic approaches. These studies demonstrate that PcrA monomers possess robust directional (3Ј to 5Ј) ssDNA translocation activity, in agreement with previous studies [5, 6], PcrA monomers are unable to unwind DNA duplexes as short as 18 base pairs. Just like Rep and UvrD monomers, PcrA monomers are very good ssDNA translocases, yet are ineffective as helicases in vitro
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