The reduced levels of chi recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo.
Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme and is stimulated by an 8-nucleotide element known as Chi (chi). We present a detailed biochemical characterization of a mutant RecBCD enzyme, designated RecBC1004D, that displays a reduced level of chi site recognition. Initially characterized genetically as unable to respond to the chi sequence, we provide evidence to indicate that the ability of this mutant enzyme to respond to chi is reduced rather than lost; the mutant displays about 20-fold lower chi recognition than wild-type RecBCD enzyme. Although this enzyme exhibits wild-type levels of double-stranded DNA exonuclease, helicase, and ATPase activity, its ability to degrade single-stranded DNA is enhanced 2-3-fold. The data presented here suggest that the reduced recombination proficiency of the recBC1004D strain observed in vivo results from a basal level of modification of the RecBC1004D enzyme at both chi-specific, as well as nonspecific, DNA sequences.
Arnold
D A
DA
Section of Genetics Graduate Group, University of California, Davis, California 95616, USA.
Bianco
P R
PR
Kowalczykowski
S C
SC
D000251:Adenosine Triphosphatases / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D009711:Nucleotides; D005090:Exodeoxyribonucleases; D005092:Exonucleases; D043211:Exodeoxyribonuclease V; D000251:Adenosine Triphosphatases; D004265:DNA Helicases