Mutations in a Conserved Motif Inhibit Single-stranded DNA Binding and Recombination Mediator Activities of Bacteriophage T4 UvsY Protein

Abstract

The UvsY recombination mediator protein is critical for homologous recombination in bacteriophage T4. UvsY uses both protein-protein and protein-DNA interactions to mediate the assembly of the T4 UvsX recombinase onto single-stranded (ss) DNA, forming presynaptic filaments that initiate DNA strand exchange. UvsY helps UvsX compete with Gp32, the T4 ssDNA-binding protein, for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. The goal of this study was to isolate mutant forms of UvsY protein that are specifically defective in UvsYssDNA interactions, so that the contribution of this activity to recombination processes could be assessed independent of other factors. A conserved motif of UvsY found in other DNA-binding proteins was targeted for mutagenesis. Two missense mutants of UvsY were isolated in which ssDNA binding activity is compromised. These mutants retain self-association activity, and form stable associations with UvsX and Gp32 proteins in patterns similar to wild-type UvsY. Both mutants are partially, but not totally, defective in stimulating UvsX-catalyzed recombination functions including ssDNAdependent ATP hydrolysis and DNA strand exchange. The data are consistent with a model in which UvsY plays bipartite roles in presynaptic filament assembly. Its protein-ssDNA interactions are suggested to moderate the destabilization of Gp32-ssDNA, whereas its protein-protein contacts induce a conformational change of the UvsX protein, giving UvsX a higher affinity for the ssDNA and allowing it to compete more effectively with Gp32 for binding sites.

Dynamics of Bacteriophage T4 Presynaptic Filament Assembly from Extrinsic Fluorescence Measurements of Gp32-Single-stranded DNA Interactions

“…Proper assembly of presynaptic filaments therefore requires the mediator function of UvsY protein in addition to UvsX and Gp32 (2,3). UvsX exhibits a strict requirement for UvsY in strand exchange assays performed at salt concentrations approximating physiological ionic strength (11,26,27), consistent with the co-dependence of in vivo recombination processes on UvsX and UvsY (28 -30). In the current model of T4 presynaptic filament assembly, UvsY weakens Gp32-ssDNA interactions and strengthens UvsX-ssDNA interactions, allowing UvsX filaments to nucleate onto pre-existing Gp32-ssDNA complexes, and ultimately to displace Gp32 from the lattice (27,(31)(32)(33).…”

“…7, blue line), suggesting that a slow and inefficient displacement of Gp32F from ssDNA by UvsX protein occurs in the presence of UvsY mutants. Because UvsY K58A and UvsY K58A,R60A have essentially no ssDNA binding activity in 200 mM NaCl (27,51), and because UvsY-mediated destabilization of Gp32-ssDNA interactions is independent of UvsY-Gp32 protein-protein interactions (32), the data in Fig. 8 suggest that UvsY-UvsX protein-protein interactions can promote a limited filament assembly process, presumably by strengthening UvsX-ssDNA interactions.…”

“…UvsX exhibits a strict requirement for UvsY in strand exchange assays performed at salt concentrations approximating physiological ionic strength (11,26,27), consistent with the co-dependence of in vivo recombination processes on UvsX and UvsY (28 -30). In the current model of T4 presynaptic filament assembly, UvsY weakens Gp32-ssDNA interactions and strengthens UvsX-ssDNA interactions, allowing UvsX filaments to nucleate onto pre-existing Gp32-ssDNA complexes, and ultimately to displace Gp32 from the lattice (27,(31)(32)(33). After transiently participating in presynapsis, Gp32 further stimulates DNA strand exchange by binding to the displaced single strand generated during UvsX-catalyzed D-loop formation (24).…”

“…In vivo, T4 homologous recombination events are equally dependent on functional UvsX and UvsY (28,29,49,50). This co-dependence is recreated in vitro by performing UvsX-catalyzed ssDNA-dependent ATPase and DNA strand exchange reactions at moderately high salt concentrations, wherein the reactions are strictly UvsY-dependent in the presence of Gp32 (11,26,27). For this reason all Gp32F-ssDNA disruption studies were performed at the moderately high salt concentration of 200 mM NaCl, wherein UvsY mediator effects are maximized.…”

“…UvsY-ssDNA Interactions Are Required for Normal Presynaptic Filament Assembly-Two UvsY missense mutants, UvsY K58A and UvsY K58A,R60A , exhibit dramatically decreased ssDNA binding ability, whereas retaining proper self-and hetero-protein associations (27,51). We tested the abilities of these mutants to replace wild-type UvsY in the kinetic competition assay for presynaptic filament assembly in 200 mM NaCl (Fig.…”