Abstract
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.
Highlights
The RecBCD enzyme is a 330-kDa protein composed of three subunits, the products of the recB, recC, and recD genes (1, 2)
This assay provides another measure of recognition, and more importantly, this assay provides a potential means of separating recognition from the modification of nuclease activity at that is required to detect -specific ssDNA fragments
In vivo data strongly imply a complete absence of recognition (24, 25), our experiments reveal that the RecBC1004D enzyme retains the ability to recognize and respond to in vitro, at one-twentieth the efficiency of the wild-type enzyme, and suggest that it may respond to weakly acting non-canonical “-like” sequences in vivo
Summary
Chemicals and Buffers—All solutions were made using Barnstead NANOpure water and reagent-grade chemicals. Smith (Hutchinson Cancer Research Center, Seattle, WA) These strains bear a chromosomal deletion from thyA to argA (which includes the recB, recC, and recD genes) and contain plasmid derivatives encoding the RecBCD or RecBC1004D enzymes (24, 26). The specific activity of the mutant enzyme preparation was determined to be 3.8 ϫ 105 nuclease units/mg of total protein, measured as described by Eichler and Lehman (3). This is comparable to wild-type RecBCD enzyme for which a specific activity of 2.4 ϫ 105 units/mg of protein was measured. SSB protein was purified from E. coli strain RLM727 as described (30) and concentration determined using ⑀ ϭ 3.0 ϫ 104 MϪ1 cmϪ1. Assays were performed as described under “Experimental Procedures” for both the wild-type and mutant enzymes in parallel trials
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