INTERACTION OF RECBCD ENZYME WITH DNA AT DOUBLE-STRAND BREAKS PRODUCED IN UV-IRRADIATED ESCHERICHIA-COLI - REQUIREMENT FOR DNA END PROCESSING

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vi vo. We found that this activity decreased strongly when cells were irr adiated with UV light (135 J/m(2)). The activity decrease was seen by an increase in survival of phage T4 2(-) of about 200-fold (phage T4 2 (-) has defective duplex DNA end-protecting gene 2 protein). The activ ity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentati on occurred as demonstrated by pulsed-field gel electrophoresis. In ac cord with previous observations, it was concluded that the RecBCD enzy me is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction o r permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb(+)) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wil d-type cells the tails are effectively removed by single-strand-specif ic DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By thi s, tailed DNA ends are processed to entry sites for RecBCD, It is prop osed that end blunting functions to direct DNA ends into the RecABCD p athway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.