Studies of the T4 recombination system have provided insights on recombination mechanisms that are highly relevant to HR and HDR processes in cellular organisms including eukaryotes. Work with T4 UvsY protein has helped to define the roles that recombination mediator proteins play in promoting presynaptic filament assembly and in the trafficking of recombination proteins (SSB, RMP, and recombinase) on ssDNA that occurs during the early stages of recombination and homology-directed DNA repair processes. It is clear that the UvsY model for assembly of recombinase filaments on ssDNA covered with ssDNA-binding protein is highly conserved [24
], including in human beings where at least three classes of proteins with UvsY-like mediator activity participate in genome stability pathways. These include Rad52, the human Rad51 paralogs Rad51B, Rad51C, Rad51D, Xrcc2, and Xrcc3, and breast cancer susceptibility gene Brca2 [97
]. Details of T4 presynaptic filament assembly and dynamics, such as ssDNA hand-offs and dynamic instability, suggest mechanisms that may be used by recombination machineries in many organisms to capture recombinagenic ssDNA, perform strand exchange, and pass the intermediates on to other repair enzymes such as the replicative components of HDR pathways.
Recent biochemical and structural studies of UvsX recombinase shed light on its mechanism and relationship to other recombinases of the RecA/Rad51 superfamily. The observation that ssDNA-binding by UvsX allosterically regulates the enzyme's affinity for homologous vs. non-homologous dsDNA at a second site is an important breakthrough [66
]. The sensitive fluorescence assay developed for this study represents an excellent opportunity to explore how micro-heterology affects homologous pairing, as well as the similarities and differences between pairing mechanisms used by recombinases from various organisms. The X-ray crystal structure of UvsX and its modeling in EM filament structures shows that UvsX shares the same extended filament structure in its active form as do E. coli
and yeast filament structures (Gajewski, S., M.R. Webb, V. Galkin, E.H. Egelman, K.N. Kreuzer, and S.W. White: Crystal structure of the phage T4 recombinase UvsX and its functional interation with the T4 SF2 helicase UvsW, unpublished). The observation that UvsX appears to share the lysine bridges found at the active site of E. coli
RecA-DNA places UvsX mechanistically closer to prokaryotic than to eukaryotic recombinases, at least in this detail. Opportunities for structure-driven mutagenesis and mechanistic studies, as well as for evolutionary studies, of UvsX will surely follow from this important structure.
The T4 field pioneered studies of helicases in recombination, which are now known to be pervasive regulators of recombination and HDR metabolism in all organisms [100
]. The biochemistry of T4 helicases demonstrates the diverse ways that these enzymes can influence recombination outcomes, including both positive and negative regulation of homologous pairing and strand exchange. It is noteworthy that T4 encodes threes different helicases on its phage genome that appear to have both unique and overlapping functions in recombination. Of particular relevance is the role of helicases in channeling strand exchange reactions toward the formation of intermediates that can serve as initiators of recombination-dependent DNA replication [4
]. T4 RDR requires either Dda (for bubble-migration DNA synthesis) or Gp41/Gp59 (for semi-conservative DNA synthesis) to initiate replication via a recombination event. The biochemical role of UvsW in the RDR machine remains to be elucidated but is likely to be central given its ability to promote extensive branch migration. The coupling of recombination to replication is fundamental for DNA repair and genome stability in all organisms. Eukaryotic DNA helicases/translocases such as Rad54, Srs2 and others are known to play important roles in processing recombination intermediates, either for regulatory purposes or to facilitate access of downstream DNA replication and repair enzymes to the products of strand exchange [10
]. The T4 helicases offer an excellent opportunity to study more about the mechanism of recombination/replication coupling, the findings of which will directly inform studies of genome stability mechanisms in cellular organisms including humans.