Bacillus subtilisRecU protein cleaves Holliday junctions and anneals single-stranded DNA

Abstract

Bacillus subtilis RecU protein is involved in homologous recombination, DNA repair, and chromosome segregation. Purified RecU binds preferentially to three- and four-strand junctions when compared to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) (≈10- and ≈40-fold lower efficiency, respectively). RecU cleaves mobile four-way junctions but fails to cleave a linear dsDNA with a putative cognate site, a finding consistent with a similar genetic defect observed for genes classified within the ε epistatic group (namely ruv A, rec D, and rec U). In the presence of Mg 2+ , RecU also anneals a circular ssDNA and a homologous linear dsDNA with a ssDNA tail and a linear ssDNA and a homologous supercoiled dsDNA substrate. These results suggest that RecU, which cleaves recombination intermediates with high specificity, might also help in their assembly.

Interaction of Branch Migration Translocases with the Holliday Junction-resolving Enzyme and Their Implications in Holliday Junction Resolution

“…4A). The sequence recognized by RecU on this arm of synthetic HJ-J3 was 5Ј-GG2CT-3Ј that is in good agreement with the previously published consensus recognition site for RecU (36). No symmetric cleavage of HJ-J3 DNA was observed in the denaturing gel (Fig.…”

“…We previously observed that RecU cleavage was often uncoupled in a synthetic HJ if the RecU consensus recognition sequence (5Ј-(G/T)G2C(A/C)-3Ј) is not present in the two opposite arms, and that the absence of RecU impairs chromosomal segregation (32,36). When the cleavage of HJ-J3 DNA by RecU was analyzed on denaturing PAGE, preferentially a nick on one arm (arm 4) was observed (Fig.…”

“…The accuracy of the constructions was verified by DNA sequencing. Plasmid-borne recU or its variants (recUR31A, recUE36A, and recUY80A) and ruvB gene were previously described (29,30,36,37).…”

“…The radiolabeled HJ-jbm6 was assembled from four 40-nucleotide oligonucleotides and gel-purified. It contains a 13-bp homologous core (36).…”

“…Proteins-RecU, RecUE36A, RecUR31A, RecUY80A, and RuvB were purified, and their concentrations were determined as previously described (23,29,30,36,37). Cells overexpressing the RuvA protein were harvested and resuspended in buffer A (50 mM Tris-HCl, pH 8.5, 1 mM EDTA, 10% glycerol) containing 100 mM KCl and lysed by sonication.…”