The PcrA helicase shows considerable homology to both E. coli UvrD and Rep, all three proteins being members of helicase superfamily I. We have overexpressed , purified, crystallised, and biochemically characterised B. stearothermophilus PcrA.

Crystallisation/Structure Determination

Crystals of the protein were obtained and the structure solved at 2.5 Å resolution (Subramanya et al., 1996). The protein comprises two domains with a deep cleft running between them. The ATP-binding site is formed by motifs that are conserved across the entire 3'-5' helicase superfamily. Unexpectedly, we found that the two regions of the protein were structurally homologous to RecA. Comparison between the ATP-binding site of RecA and the helicase reveals that all of the key residues in the active site of RecA are conserved in space in the helicase active site.

Substrate and product complexes of PcrA helicase with a DNA substrate have also been solved which imply that the enzyme operates by an inchworm mechanism.

PcrA Biochemistry

Biochemistry

PcrA is able to couple the hydrolysis of a broad range of nucleotides to a 3'-5' helicase strand separation reaction. The enzyme shows a specificity for the DNA substrate in gel mobility assays with the preferred substrate being one containing both single and double stranded regions of DNA. In contrast to Rep and UvrD from E. coli, we do not see any evidence for dimerisation of the enzyme using gel filtration, or by crosslinking in the presence of combinations of Mg²⁺, nucleotides and DNA. Moreover, k_cat for ATP hydrolysis is constant over a large range of protein concentrations. Therefore, the protein appears to be monomeric under all conditions tested, including in the structure of two crystal forms of PcrA.

Interactions with effector proteins

Ribosomal protein L3

During the purification of mutant PcrA proteins a low molecular weight protein co-purified with one of our mutant proteins. We identified this protein, by N-terminal protein sequencing, as the E.coli ribosomal protein L3. Further investigation revealed that purified L3 protein affected the helicase activity of wild type (wt) PcrA. L3 enhances the ability of wt PcrA to displace short oligonucleotides annealed to M13 ssDNA, although L3 by itself has no ATPase activity and does not stimulate the ATPase activity of PcrA. Furthermore, it has no significant unwinding activity on short oligonucleotides annealed to M13 ssDNA. However, as shown by electrophoretic mobility shift assays, it is able to bind either ds or ssDNA. We examined the mechanism by which ribosomal protein L3 stimulates the unwinding activity of PcrA. L3 stimulates the helicase activity of PcrA in a substrate-independent manner and enhances co-operative binding of PcrA onto its DNA substrate as shown by electrophoretic mobility shift assays.

We have cloned and over-expressed the ribosomal protein L3 from B. stearothermophilus. We are now trying to purify L3 in order to obtain large quantities of pure protein for crystallisation trials. Emphasis will be given to the crystallisation of L3 alone and also to the crystallisation of the L3-PcrA complex. Further biochemical analysis will also be performed to elucidate in more detail the stimulatory effect of L3 upon the helicase activity of PcrA.