Chi Hotspot Activity in Escherichia coli Without RecBCD Exonuclease Activity: Implications for the Mechanism of Recombination

摘要

The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCDnenzyme, a complex nuclease and DNA helicase regulated by Chi sites (59-GCTGGTGG-39). During itsnunwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions,ndepending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching ofnnucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recCnmutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but lossnof detectable nucleolytic degradation as judged by the growth of mutant T4 and l phages and by assay ofncell-free extracts. We conclude that RecBCD enzyme’s nucleolytic degradation of DNA is not necessary fornintracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. Wendiscuss the bearing of these results on current models of RecBCD pathway recombination.