Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and p roductive loading of specific enzymes onto single-stranded DNA (ssDNA) duri ng phage RDR processes. The T4 recombination mediator protein, uvsY, is nec essary for the proper assembly of the T4 presynaptic filament (uvsX recombi nase cooperatively bound to ssDNA), leading to the recombination-primed ini tiation of leading strand DNA synthesis. In the lagging strand synthesis co mponent of RDR, replication mediator protein gp59 is required for the assem bly of gp41,the DNA helicase component of the T4 primosome, onto lagging st rand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homo logous recombination events to the initiation of T4 RDR. UvsY promotes pres ynaptic filament formation on 3 ' ssDNA-tailed chromosomes, the physiologic al primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of d ouble-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX b ound to the invading strand, effectively preventing primosome assembly ther e. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 rec ombination/replication mediator proteins guards against antirecombination a ctivity of the helicase component and ensures that recombination intermedia tes formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservati ve, we present biochemical evidence that a conservative "bubble migration" mode of RDR could play a role in lesion bypass by the T4 replication machin ery.