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 recUDelta1-32 (which initiates at Met33 and encodes RecUDelta1-32) 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.ATPgammaS.Mg(2+) fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUDelta1-32 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.
Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Cantoblanco, Madrid, Spain.
Begoña
B
Carrasco
Cristina
C
Cañas
Gary J
GJ
Sharples
Juan C
JC
Alonso
Silvia
S
Ayora
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology* / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020090:Chromosome Segregation; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D045565:Holliday Junction Resolvases / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017384:Sequence Deletion
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D045565:Holliday Junction Resolvases