Specific recognition of the viral genome is an essential step in the assembly of most viruses, including tailed bacteriophages. Errors in this mechanism lead to horizontal transfer of cellular genes (transduction) playing a major role in bacteria evolution and adaptation. In numerous phages including SPP1, genome packaging is initiated by recognition of the pac sequence within a viral DNA concatemer by the small subunit of the terminase (TerS). Pac is subsequently cut by the terminase large subunit (TerL) generating a DNA free end that is the starting point for the unidirectional packaging of viral DNA into the phage procapsid. In spite of its critical role to ensure specificity of phage DNA packaging, the molecular mechanism of pac recognition and cleavage remains very poorly understood.
In this study we used a plasmid minimal system encoding SPP1 pac, TerS and TerL that mimics specific pac recognition and its auto-regulated cleavage in Bacillus subtilis, the SPP1 host. The pac sequence can be subdivided in three sub-regions: pacC, bearing the cleavage site, surrounded by TerS binding sites pacL and pacR. These sequences were degenerated to determine their role in pac cleavage.
The endonucleolytic cleavage that initiates the packaging reaction is shown to occur centered at a defined position in pacC with a 5 bp precision but to not require a specific sequence. A pacL 76 bp region that interacts with TerS and a poly-adenine tract within pacR are necessary for pac cleavage. Our results support a model in which the DNA-binding domains of TerS nonamer ring(s) bind at several positions of pacL and interact with the pacR polyA sequence. This nucleoprotein architecture creates the structural context for recruitment of TerL to cleave accurately within pacC. This complex protein-DNA interaction is proposed to be necessary for highly specific, controlled and unidirectional packaging of phage DNA.