Structure and mechanism of the phage T4 recombination mediator protein UvsY

Abstract

The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY-ssDNA interaction occurs within the assembly via two distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNAgp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNAbinding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA-UvsX filament.homologous recombination | structural modification | DNA binding | DNA architecture | crystallography H omologous recombination (HR) involves the exchange of strands between homologous DNA molecules and has fundamental roles in double-stranded DNA (dsDNA) break repair, the rescue of stalled replication forks, and recombination-dependent replication (1). Defects in HR are associated with genetic instability, chromosomal abnormalities, and cancer (1). HR is performed by an ATP-dependent recombinase, RecA in prokaryotes and Rad51 in eukaryotes, that creates a filament with approximately sixfold helical symmetry. The filament first binds single-stranded DNA (ssDNA) and then samples incoming dsDNA to search for homology and promote the exchange reaction (2, 3). Key insights into the mechanism have been provided by structural (4) and dynamics (5, 6) studies of the HR filament. ssDNA-binding proteins, RPA in eukaryotes and SSB in prokaryotes, protect the ssDNA from nucleases and remove secondary structures during HR, but they also block the binding of the recombinase (3). This obstacle is overcome by the recombination mediator proteins (RMPs) (7), Rad52 in eukaryotes and RecOR in prokaryotes, that stimulate the exchange of the ssDNA-binding proteins for the recombinase.Phage T4 is able to process DNA in isolation from the host Escherichia coli by encoding all of the necessary DNA metabolic proteins. The T4 UvsXYW system encodes the core HR machinery (8) comprising UvsX (the recombinase), UvsW (the SF2 remodeling helicase), and UvsY (the RMP). Together with the T4 ssDNA-binding protein gp32 (9, 10), these three proteins can efficiently perform HR in an in vitro reconstituted system (11). UvsY is a 15.8-kDa protein with properties that are consistent with its rol...

In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

“…The structures of both the gp59 and UvsY proteins have been determined by x-ray crystallography (Mueser et al , 2000; Gajewski et al , 2016), and their detailed modes of action have been analyzed by physicochemical techniques (Bleuit et al , 2001; Zhang et al , 2005; Liu et al , 2006, 2013; Xu et al , 2010; Branagan et al , 2012, 2014; Benkovic and Spiering, 2017). These small proteins readily oligomerize: gp59 to form hexamers and UvsY to form heptamers.…”

Elucidating Recombination Mediator Function Using Biophysical Tools

“…RMP UvsY in T4 adopts a heptameric open barrel conformation [ 80 ] which wraps ssDNA upon binding. UvsY further forms a complex with gp32-ssDNA in which gp32 and UvsY interact with a 1:1 stoichiometry [ 81 , 82 ].…”

“…Gajewski and co-workers examined the formation of the ternary complex of bacteriophage T4 recombination mediator UvsY, single-stranded binding protein gp32 and ssDNA utilizing SPR ( Figure 3 A, [ 80 ]). C-terminal deletion mutant of gp32, gp32ΔC240-301, does not support formation of ternary complex, whereas the N-terminal deletion mutant, gp32ΔN1-21 can still form ternary complexes.…”

“…No complex containing both RecO and RecF has ever been detected and recent results lead to questions about the status of RecF as an RMP. ( B ) Bacteriophage T4 RMP—UvsY—adopts a multimeric ring-like shape [ 80 ]. Multimers of UvsY then destabilize gp32 filaments by bending the DNA and exchange gp32 with UvsX recombinase molecules via direct interaction, thereby stabilizing UvsX presynaptic filaments [ 80 , 81 , 82 ].…”

“…( B ) Bacteriophage T4 RMP—UvsY—adopts a multimeric ring-like shape [ 80 ]. Multimers of UvsY then destabilize gp32 filaments by bending the DNA and exchange gp32 with UvsX recombinase molecules via direct interaction, thereby stabilizing UvsX presynaptic filaments [ 80 , 81 , 82 ]. ( C ) Eukaryotic RMP—RAD52—forms undecamer rings [ 83 ] which can promote RAD52-depedent DNA annealing and RAD51 D-loop formation [ 84 , 85 , 86 ].…”