Facilitated Loading of RecA Protein Is Essential to Recombination by RecBCD Enzyme

Although the RecB2109CD 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 produci...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-04, Vol.275 (16), p.12261-12265
Main Authors: Arnold, Deana A., Kowalczykowski, Stephen C.
Format: Article
Language:English
Subjects:
DNA, Single-Stranded - metabolism
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Escherichia coli Proteins
Exodeoxyribonuclease V
Exodeoxyribonucleases - genetics
Exodeoxyribonucleases - metabolism
exonuclease
Models, Chemical
Molecular Sequence Data
Mutagenesis
Phenotype
Rec A Recombinases - metabolism
RecBCD enzyme
Recombination, Genetic
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Summary:Although the RecB2109CD 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 χ-modified wild-type RecBCD enzyme. However, we further show that the RecB2109CD 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 χ-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.275.16.12261