Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination

Abstract

Bacteriophage T4 homologous recombination events are promoted by presynaptic filaments of UvsX recombinase bound to single-stranded DNA (ssDNA). UvsY, the phage recombination mediator protein, promotes filament assembly in a concentration-dependent manner, stimulating UvsX at stoichiometric concentrations but inhibiting at higher concentrations. Recent work demonstrated that UvsX-H195Q/A mutants exhibit decreased ssDNA-binding affinity and altered enzymatic properties. Here, we show that unlike wild-type UvsX, the ssDNA-dependent ATPase activities of UvsX-H195Q/A are strongly inhibited by both low and high concentrations of UvsY protein. This inhibition is partially relieved by UvsY mutants with decreased ssDNA-binding affinity. The UvsX-H195Q mutant retains weak DNA strand exchange activity that is inhibited by wild-type UvsY, but stimulated by ssDNA-binding compromised UvsY mutants. These and other results support a mechanism in which the formation of competent presynaptic filaments requires a hand-off of ssDNA from UvsY to UvsX, with the efficiency of the hand-off controlled by the relative ssDNA-binding affinities of the two proteins. Other results suggest that UvsY acts as a nucleotide exchange factor for UvsX, enhancing filament stability by increasing the lifetime of the high-affinity, ATP-bound form of the enzyme. Our findings reveal new details of the UvsX/UvsY relationship in T4 recombination, which may have parallels in other recombinase/mediator systems.

Presynaptic filament dynamics in homologous recombination and DNA repair

“…Next, UvsY alters ssDNA structure in ways that destabilize Gp32-ssDNA interactions while promoting UvsX-ssDNA filament nucleation (Sweezy and Morrical 1999, Pant et al 2008, Liu et al 2006a, Farb and Morrical 2009b). The ssDNA structural transitions appear to involve different wrapped states of ssDNA around UvsY hexamers (Xu et al 2010, Pant et al 2008).…”

“…Finally, UvsX is loaded onto a UvsY-Gp32-ssDNA intermediate with concomitant displacement of Gp32 (Liu et al 2006b). Thus presynaptic filament assembly involves a series of ssDNA handoff transactions that is best described by the progression Gp32-ssDNA + UvsY → UvsY-Gp32-ssDNA → UvsY-Gp32-ssDNA* + UvsX → UvsX-UvsY-ssDNA + Gp32, where UvsY-Gp32-ssDNA and UvsY-Gp32-ssDNA* represent different wrapped conformations of ssDNA (Farb and Morrical 2009b, Xu et al 2010). …”

“…UvsX generates both ADP and AMP as ATP hydrolysis products (Formosa and Alberts 1986b, Farb and Morrical 2009a), and UvsY specifically promotes ADP generation and suppresses further ADP hydrolysis to AMP (Farb and Morrical 2009b). Thus, it was proposed that UvsY could also enhance presynaptic filament stability by acting as an ADP/ATP exchange factor, which would increase the average lifetime of the high affinity form of UvsX on the ssDNA (Farb and Morrical 2009b).…”

“…Thus, it was proposed that UvsY could also enhance presynaptic filament stability by acting as an ADP/ATP exchange factor, which would increase the average lifetime of the high affinity form of UvsX on the ssDNA (Farb and Morrical 2009b). …”

“…Mutations in a key allosteric switch residue of UvsX protein, UvsX-H195A and H195Q, display reduced ssDNA-binding and strand exchange activities (Farb and Morrical 2009b). Interestingly, UvsY mutants with reduced ssDNA-binding affinity partially rescue enzymatic activities of the UvsX mutants (Bleuit et al 2001, Farb and Morrical 2009b).…”