The structure of bacteriophage T5 injection stop signal.

Escherichia coli bacteriophage T5 differs from most phages in that it injects its genome in two steps: First Step Transfer, FST, corresponding to leftmost 7.9% of the genome and Second Step Transfer, SST, corresponding to the remainder of the genome. Expression of genes A1 and A2 is required for SST. DNA injection stops at a site known as the injection stop signal (iss) which is a cis acting site located in the large untranslated region of the Left Terminal Repeat (LTR). The iss region is extremely complicated with many repeats, inverted repeats and palindromes. This report compares the iss regions of 25 T5-like phages and shows that all have a common conserved structure including a series of 8 DnaA boxes arranged in a highly specific manner; reminiscent of the origin of replication (oriC) of E. coli. DnaA protein, which binds to DnaA boxes, is a mostly membrane bound. A new, radically different, mechanism of T5 2-step injection is proposed whereby injecting T5 DNA stops at the plasma membrane due to the binding of the iss DnaA boxes to membrane-bound DnaA protein. Injection of the SST continues later via the combined action of the A1 and A2 proteins which cleave the FST DNA at a site upstream (right) of the iss region, thereby liberating it. They also cleave the incoming SST DNA at the same site on the RTR thus facilitating circularisation of one complete genome via the cohesive ends. Circle formation protects the T5 DNA from the degradative action of the RecBCD nuclease and eventually leads to rolling circle DNA replication.