Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme.
Although the RecB(2109)CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination. Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3'-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion. Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the chi-modified wild-type RecBCD enzyme. However, we further show that the RecB(2109)CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele. Our findings argue that the facilitated loading of RecA protein by the chi-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.
Arnold
D A
DA
Section of Genetics Graduate Group, University of California, Davis, California 95616, USA.
Kowalczykowski
S C
SC
D004277:DNA, Single-Stranded / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli