We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo. We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzym...
We have analyzed the transfer kinetics of recA protein from one polynucleotide to another by monitoring the change in fluorescence of a modified single-stranded M13 DNA, referred to as etheno M13 DNA, that accompanies recA protein dissociation. The observed rate of transfer is dependent on the concentration of competitor polynucleotide, polythymidy...
The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of...
We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4 ,6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the R...
In Escherichia coli, chi (5 -GCTGGTGG-3 ) is a recombination hotspot recognized by the RecBCD enzyme. Recognition of chi reduces both nuclease activity and translocation speed of RecBCD and activates RecA-loading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain t...
The RecA142 protein, in which valine is substituted for isoleucine-225, is defective for genetic recombination in vivo and for DNA strand exchange activity in vitro under conventional growth and reaction conditions respectively. However, we show that mildly acidic conditions restore both the in vitro DNA strand exchange activity and the in vivo fun...
We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stoppe...
