Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY.
Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.
Department of Physics, Northeastern University, Boston, Massachusetts 02115, USA.
Kiran
K
Pant
Leila
L
Shokri
Richard L
RL
Karpel
Scott W
SW
Morrical
Mark C
MC
Williams
D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D011995:Recombination, Genetic; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D012965:Sodium Chloride; D014357:Trypsin