pH-dependent Activities and Structural Stability of Loop-2-anchoring Helix of RadA Recombinase from Methanococcus voltae
D. E. C. S. Rao and Yu Luo
Affiliation: Department of Biochemistry, University of Saskatchewan, 2D01 Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5.
RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also
includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in
the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous doublestranded
DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues,
we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular
mechanism of homologous strand exchange. Here we show this protein’s ATPase and strand exchange activities are
minimal at pH 6.0. Interestingly, MvRadA’s pH dependence is similar to the properties of human Rad51 but dissimilar to
that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase-
inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5
implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further
suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously
observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand
double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent
activities of MvRadA.
Keywords: ATPase, conformational change, DNA strand exchange, DMC1, homologous recombination, RadA, Rad51, RecA.
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