ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature

Abstract

RecD is a 5¢ fi 3¢ helicase motor protein. The primary sequence contains the characteristic seven conserved motifs (I, Ia, II, III, IV, V, and VI) of the superfamily 1 (SF1) group of DNA helicases [1] (Fig. 1). In Escherichia coli, RecD displays ssDNA-dependent ATPase and helicase activity in vitro [2,3]. In vivo, it functions as a component of the RecBCD complex (also known as exonuclease V) that is involved in DNA repair and recombination in many bacteria [4]. RecBCD is a highly processive helicase ⁄ nuclease enzyme with dual motor activity, in which RecB and RecD subunits, with their respective (3¢ fi 5¢) and (5¢ fi 3¢) polar movement, translocate the enzyme along the anti-parallel strands of dsDNA. DNA unwinding by helicase activity is accompanied by degradation of the strands until the enzyme encounters the recombination hotspot v (chi) sequence (5¢-GCTGGTGG-3¢). This changes the nuclease property of the enzyme, leading to the generation of 3¢-extended ssDNA and loading of RecA onto the DNA for homologous pairing and DNA strand exchange, producing recombination intermediates [5]. Interestingly, however, RecBC alone is proficient for recombination and repair of DNA, and recD-inactivated mutants of E. coli do not show any growth defects [6,7]. Thus, the contribution of the RecD subunit is thought to be of less significance in vivo. Remarkably, RecD inactivation leads to the loss of exonuclease V activity in cells, despite the fact that the only nuclease catalytic center of RecBCD complex lies in the RecB subunit [8]. Hence, a role for RecD in regulating the nuclease activity of RecBCD has been advocated. Recently, using ATP RecD is essential for growth at low temperature in the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro, with the ability to unwind short DNA duplexes containing only 5¢ overhangs or forked ends. Five of the mutant proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in motif III) and R660A (in motif VI) completely lost both ATPase and helicase activities. Three other mutants, T259A in motif Ia, R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity, but had no helicase activity. While all RecD proteins had DNAbinding activity, the mutants of motifs IV and V displayed reduced binding, and the motif II mutant showed a higher degree of binding to ssDNA. Significantly, only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4°C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.Abbreviations ABM, Antarctic bacterial medium; ATPc-S, adenosine 5¢-O-(thiophosphate); EMSA, electrophoretic gel mobility shift assay; SF1, superf...

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

“…Although MtRecD can bind linear duplex having 5Ј-or 3Ј overhangs with nearly equal efficiency, unwinding of the 5Ј overhang substrate was more efficient than unwinding of substrates containing 3Ј overhangs. These results differ from those reported for D. radiodurans RecD1, Pseudomonas syringae RecD, and UvrD helicase (64,67,68). MtRecD also exhibited ATP binding as well as ssDNA-dependent ATPase activity, consistent with the activities of E. coli RecD and D. radiodurans RecD2 (52, 64).…”

Insight into the Roles of Helicase Motif Ia by Characterizing Fanconi Anemia Group J Protein (FANCJ) Patient Mutations

“…In Pseudomonas syringae, RecD helicase T259A mutant in motif Ia ( 258 PTGKAAAR 265 ) retains ssDNA binding function. However, the protein displays reduced ATPase activity (72%) and lacks DNA unwinding activity in vitro, but the T259A mutant is able to complement the growth defect of a recDdisrupted strain of P. syringae (53). Retention of the biological activity suggests that the uncoupled ATPase activity in the mutant protein might have other significance.…”

All Three Subunits of RecBCD Enzyme Are Essential for DNA Repair and Low-Temperature Growth in the Antarctic Pseudomonas syringae Lz4W

“…For genetic complementation analysis, plasmids were constructed for the expression of recB , recC or recD individually or in combinations, using the broad-host-range plasmid pGL10 as described earlier [52]. The primers used for PCR amplification of the genes are enlisted in Supporting Information S1.…”

“…The His-tagged ORF of recB gene was then cleaved out from pETB as ( Xba I- Sac I) fragment and ligated into pGL10 to generate pGB. The pGD, which produced C-terminally 6×His-tagged RecD, has been described earlier [52]. For the construction of pGCBD which expressed all three subunits of RecBCD Ps , 9.215 kbp DNA containing the three reading frames of recCBD operon of P. syringae was amplified by PCR using FCNH1 and RDS1 primers.…”