STRAND SPECIFICITY OF NICKING OF DNA AT CHI-SITES BY RECBCD ENZYME - MODULATION BY ATP AND MAGNESIUM LEVELS

RecBCD enzyme is essential for the major pathway of homologous recombi nation of linear DNA in Escherichia coli. It is a potent nuclease and helicase and, during its unwinding of double-stranded DNA, makes singl e-strand scissions in the vicinity of Chi recombination hot spots. We report here that both the strand that is cut and the position of the c uts relative to Chi depended on the ATP to Mg2+ ratio. With ATP in exc ess, Chi-dependent nicks occurred, as we have previously reported, fou r to six nucleotides to the 3'-side of the Chi octamer (5'-GCTGGTGG-3' ) and were detected only on the strand bearing that sequence. Three di fferences were seen with Mg2+ in excess. 1) Chi-dependent 3'-ends were produced on the GCTGGTGG-containing strand closer to and within the C hi octamer, 2) Chi-dependent cuts occurred on the complementary DNA st rand, 3) RecBCD enzyme destroyed the 3'-terminated strand of DNA from its entry point up to the vicinity of the Chi site, as others have pre viously reported. We show here that, with Mg2+ in excess, the enzyme c ontinued to travel along DNA, after encountering a Chi site, releasing both strands of the DNA distal to Chi as single strands. We discuss p otential biological consequences of these two modes of RecBCD enzyme-C hi interaction.