THE LARGEST (70 KDA) PRODUCT OF THE BACTERIOPHAGE-T4 DNA TERMINASE GENE-17 BINDS TO SINGLE-STRANDED-DNA SEGMENTS AND DIGESTS THEM TOWARDS JUNCTIONS WITH DOUBLE-STRANDED DNA

Bacteriophage terminases are oligomeric multifunctional proteins that bind to vegetative DNA, cut it and, together with portal proteins, tra nslocate the DNA into preformed heads. Most terminases are encoded by two partially overlapping genes. In phage TS they are genes 16 and 17. We have shown before that the larger of these, gene 17, can yield, in addition to a full-length 70 kDa product, several shorter peptides. A t least two of these, gene product (gp) 17' and gp17 '', are initiated in the same reading frame as the 70 kDa gp17 from internal ribosome b inding sites. Most of the shorter gp17 s contain predicted ATPase moti fs, but only the largest (70 kDa) peptide has a predicted single-stran ded DNA binding domain. Here we describe the DNA binding and cutting p roperties of the purified 70 kDa protein, expressed from two different clones containing gene 17 but no other T4 gene. Epitope-specific anti bodies, which recognize several different gene 17 products in extracts of induced clones or of T4-infected cells, precipitate the purified 7 0 kDa gp17. When Mg2+ is chelated by EDTA this 70 kDa protein binds to single-stranded DNA, preferentially to junctions of single-and double -stranded DNA segments. It does not bind to blunt-ended double-strande d DNA. When Mg2+ is present the purified 70 kDa gp17 digests single-st randed segments preferentially up to junctions with double-stranded DN A. A 70 kDa gp17 from a P379L temperature sensitive (ts) mutant, which has lost the nuclease and ATPase activities, retains the single-stran ded DNA binding activity. Taken together with earlier findings these r esults support a model for packaging of T4 DNA from single-stranded re gions in recombinational or replicative intermediates, which occur at nearly random positions of the genome. This mechanism may be an altern ative to site-specific initiation of packaging proposed by other inves tigators. (C) 1998 Academic Press Limited.