The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec⁺ cells, only the full-length RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible N-terminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB·ATPγS·Mg²⁺ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecA-mediated DNA strand exchange. This study shows that the flexible N-terminus of RecU is essential for protein activity.