Stopped-Flow Studies of the Kinetics of Single-Stranded DNA Binding and Wrapping around the Escherichia coli SSB Tetramer

Abstract

We have examined the kinetic mechanism for binding of the homotetrameric Escherichia coli SSB protein to single-stranded oligodeoxynucleotides [(dT)₇₀ and (dT)₃₅] under conditions that favor the formation of a fully wrapped ssDNA complex in which all four subunits interact with DNA. Under these conditions, a so-called (SSB)₆₅ complex is formed in which either one molecule of (dT)₇₀ or two molecules of (dT)₃₅ bind per tetramer. Stopped-flow studies monitoring quenching of the intrinsic SSB Trp fluorescence were used to examine the initial binding step. To examine the kinetics of ssDNA wrapping, we used a single-stranded oligodeoxythymidylate, (dT)₆₆, that was labeled on its 3‘-end with a fluorescent donor (Cy3) and on its 5‘-end with a fluorescent acceptor (Cy5). Formation of the fully wrapped structure was accompanied by extensive fluorescence resonance energy transfer (FRET) from Cy3 to Cy5 since the two ends of (dT)₆₆ are in close proximity in the fully wrapped complex. Our results indicate that initial ssDNA binding to the tetramer is very rapid, with a bimolecular rate constant, k_1,app, of nearly 10⁹ M^-1 s^-1 in the limit of low salt concentration (-¹. The implications of this rapid binding and wrapping reaction are discussed.