The RecU Holliday junction resolvase acts at early stages of homologous recombination.
Homologous recombination is essential for DNA repair and generation of genetic diversity in all organisms. It occurs through a series of presynaptic steps where the substrate is presented to the recombinase (RecA in bacteria). Then, the recombinase nucleoprotein filament mediates synapsis by first promoting the formation of a D-loop and later of a Holliday junction (HJ) that is subsequently cleaved by the HJ resolvase. The coordination of the synaptic step with the late resolution step is poorly understood. Bacillus subtilis RecU catalyzes resolution of HJs, and biochemical evidence suggests that it might modulate RecA. We report here the isolation and characterization of two mutants of RecU (recU56 and recU71), which promote resolution of HJs, but do not promote RecA modulation. In vitro, the RecU mutant proteins (RecUK56A or RecUR71A) bind and cleave HJs and interact with RuvB. RecU interacts with RecA and inhibits its single-stranded DNA-dependent dATP hydrolysis, but RecUK56A and RecUR71A do not exert a negative effect on the RecA dATPase and fail to interact with it. Both activities are important in vivo since RecU mutants impaired only in RecA interaction are as sensitive to DNA damaging agents as a deletion mutant.
Cañas
Cristina
C
Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, C/Darwin 3, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Carrasco
Begoña
B
Ayora
Silvia
S
Alonso
Juan C
JC
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045566:DNA, Cruciform / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D045565:Holliday Junction Resolvases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D045566:DNA, Cruciform; D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D011938:Rec A Recombinases; D045565:Holliday Junction Resolvases; D000251:Adenosine Triphosphatases; C026600:2'-deoxyadenosine triphosphate