LACTOCOCCUS-LACTIS PHAGE OPERON CODING FOR AN ENDONUCLEASE HOMOLOGOUSTO RUVC

The function of the Lactococcus lactis bacteriophage blL66 middle time -expressed operon (M-operon), involved in sensitivity to the abortive infection mechanism AbiD1, was examined. Expression of the M-operon is detrimental to Escherichia coli cells, induces the SOS response and i s lethal to recA and recBC E. coli mutants, which are both deficient i n recombinational repair of chromosomal double-stranded breaks (DSBs), The use of an inducible expression system allowed us to demonstrate t hat the M-operon-encoded proteins generate a limited number of randoml y distributed chromosomal DSBs that are substrates for ExoV-mediated D NA degradation. DSBs were also shown to occur upstream of the replicat ion initiation point of unidirectionally theta-replicating plasmids. T he characteristics of the DSBs lead us to propose that the endonucleol ytic activity of the M-operon is not specific to DNA sequence, but rat her to branched DNA structures. Genetic and physical analysis performe d with different derivatives of the M-operon indicated that two orfs ( orf2 and orf3) are needed for nucleolytic activity. The orf3 product h as amino acid homology with the E. coli RuvC Holliday junction resolva se. By site-specific mutagenesis, we have shown that one of the amino acid residues constituting the active centre of RuvC enzyme (Glu-66) a nd conserved in ORF3 (Glu-67) is essential for the nucleolytic activit y of the M-operon gene product(s). We therefore propose that orf2 and orf3 of the M-operon code for a structure-specific endonuclease (WI-nu clease), which might be essential for phage multiplication.