In many tailed bacteriophages, DNA packaging is initiated by recognition and cleavage of a specific sequence pac by the small (TerS) and large (TerL) terminase subunits. It was previously shown that the SPP1 pac region has two sequences where TerS binds (pacR and pacL), flanking the segment where TerL cleaves the SPP1 DNA (pacC). However, the pac-specific sequences required to achieve this endonucleolytic cut were not established. Their characterization is essential to understand the underlying mechanism. We show that the pacR sequence localized within 35bp downstream of the pac cut can be extensively degenerated, including its c1 and c2 repeats, and that only a disruption of a 5-bp polyadenine tract impairs the pac cleavage. This result together with deletion analysis of pacL shows that the specific DNA sequences required for targeting the terminase for pac cleavage are considerably shorter than the large region bound by TerS. Furthermore, extensive degeneration of the 6-bp target sequence within pacC where pac cleavage occurs reveals that TerL maintains, remarkably, its precise position of cleavage. Studies with SPP1-related phages show the conservation of the cut position, irrespective of the sequence variation in pacC and in pacR or the changes in pacL-pacC distance. Mechanistically, our data are compatible with a model in which TerS interactions with part of the pacL sequence and a poly-A tract in pacR are sufficient to orient very accurately the TerL nuclease to a defined pacC position. They also demonstrate that the resulting precise cut at pacC is independent of the targeted DNA sequence.
Keywords: DNA packaging; bacteriophage; genome recognition; pac cleavage; terminase.
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