A bacteriophage T4 DNA "synapsis model" proposes that the bacteriophage T4 terminase small subunit (TerS) apposes two pac site containing dsDNA homologs to gauge concatemer maturation adequate for packaging initiation. N-terminus, C-terminus, or both ends modified fusion Ter S proteins retain function. Replacements of the TerS gene in the T4 genome with fusion genes encoding larger (18-45 kDa) TerS-eGFP and TerS-mCherry fluorescent fusion proteins function without significant change in phenotype. Co-infection and co-expression by T4 phages encoding TerS-eGFP and TerS-mCherry shows in vivo FRET in infected bacteria comparable to that of the purified, denatured and then renatured, mixed fusion proteins in vitro. FRET of purified, denatured-renatured, mixed temperature sensitive and native TerS fusion proteins at low and high temperature in vitro shows that TerS ring-like oligomer formation is essential for function in vivo. Super-resolution STORM and PALM microscopy of intercalating dye YOYO-1 DNA and photoactivatable TerS-PAmCherry-C1 fusions support accumulation of TerS dimeric or multiple ring-like oligomer structures containing DNA and gp16-mCherry in vivo as well as in vitro to regulate pac site cutting.
Keywords: DNA packaging; Gene amplification; Super-resolution microscopy; Terminase.
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