RecBC enzyme nicking at chi sites during DNA unwinding: Location and orientation-dependence of the cutting

Homologous recombination by the E. coli RecBC pathway occurs at elevated frequency near Chi sites. We reported previously that Chi induces RecBC enzyme to cleave one DNA strand-that containing the Chi sequence 5′G-C-T-G-G-T-G-G3′. We report here that the Chi-dependent cleavage occurs four, five, or six nucleotides to the 3′ side of the Chi octamer and produces nicks with 3′-OH and 5′-PO₄ groups. Chi-dependent cleavage occurs if RecBC enzyme approaches the Chi sequence from the right, but not if it approaches only from the left, during unwinding of the duplex DNA substrate. A single RecBC enzyme molecule appears to cleave the DNA and to release part of it as a single-stranded fragment. These and previous results indicate that Chi-dependent cleavage is concomitant with DNA unwinding by RecBC enzyme and provide an enzymatic basis for the orientation-dependence of Chi recombinational hotspot activity. These observations demonstrate a key step of a proposed model of recombination in which RecBC enzyme produces a potentially invasive single-stranded DNA tail extending from Chi to its left. We discuss the relation between the action of Chi sites and that of special sites enhancing eukaryotic recombination.