SINGLE-STRANDED-DNA BINDING-PROPERTIES OF THE UVSY RECOMBINATION PROTEIN OF BACTERIOPHAGE-T4

Citation
Ma. Sweezy et Sw. Morrical, SINGLE-STRANDED-DNA BINDING-PROPERTIES OF THE UVSY RECOMBINATION PROTEIN OF BACTERIOPHAGE-T4, Journal of Molecular Biology, 266(5), 1997, pp. 927-938
Citations number
41
Categorie Soggetti
Biology
ISSN journal
00222836
Volume
266
Issue
5
Year of publication
1997
Pages
927 - 938
Database
ISI
SICI code
0022-2836(1997)266:5<927:SBOTUR>2.0.ZU;2-0
Abstract
The uvsY protein is an essential component of the bacteriophage T4 gen eral recombination machinery. The properties of this 16 kDa protein in clude selective binding to ssDNA, as well as specific protein-protein interactions with other T4 recombination proteins including uvsX (gene ral recombinase) and gp32 (ssDNA-binding protein). uvsY promotes the a ssembly of uvsX-ssDNA filaments, the active species in uvsX-catalyzed DNA rearrangements, apparently by helping uvsX displace gp32 from the ssDNA. To better understand the role of uvsY in the T4 recombination s ystem, here we characterize the thermodynamic and molecular properties of the interaction of the uvsY protein with a model single-stranded p olynucleotide, epsilon DNA, which is a fluorescent, etheno-modified fo rm of random-sequence ssDNA. We have found that the binding of uvsY pr otein enhances the fluorescence of the epsilon DNA lattice and that th e maximal amount of fluorescence enhancement observed is dependent on salt concentration. In addition, we have used the epsilon DNA fluoresc ence enhancement assay to establish thermodynamic parameters of bindin g and to define some of the molecular details of uvsY-epsilon DNA inte ractions. We show that uvsY binds to epsilon DNA in a non-cooperative manner, with a binding site size of four nucleotide residues per monom er of uvsY, and that this binding is salt-sensitive and involves the d isplacement of anions from the uvsY protein. We further show that uvsY protein binds preferentially to epsilon DNA over unmodified ssDNA. Th e significance of these results is discussed in light of current model s of uvsY action in the T4 recombination system. (C) 1997 Academic Pre ss Limited.