STRUCTURAL-ANALYSIS OF DNA CLEAVED IN-VIVO BY BACTERIOPHAGE-T4 TERMINASE

Phage terminases are protein complexes that cleave concatemeric phage DNA and generate termini of the packaged DNA molecule. In phage T4, th e DNA packaging proteins gp16 and gp17 are supposed to function as ter minases. The recombinant T4 terminase proteins, upon expression in viv o from strong promoters, cleaved plasmid DNA in a sequence-independent manner. Resolution of the cleaved DNA by agarose-gel electrophoresis showed a smear throughout the lane including a fraction that was retai ned in the well [Bhattacharyya and Rao, Virology 196 (1993) 34-44]. Th e appearance of a smear in the high-M(r) region could not be explained solely on the basis of a simple random-cutting mechanism. Various hyp otheses were tested to elucidate the structure of the high-M(r) DNA. T he data show that the high-M(r) DNA did not arise either by attachment of protein(s) to DNA, or by covalent linkage of cleaved DNA molecules by a recombinational mechanism. It appears that the high-M(r) DNA aro se as a result of non-covalent linkage of plasmid DNA through single s trands. A working model for the action of T4 terminase is presented.