Stopped-flow studies of the kinetics of single-stranded DNA binding and wrapping around the escherichia coli SSB tetramer

摘要

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 conditiohs that favor the formationof 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 quenchingof the intrinsic SSB Trp fluoresence 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'-endwith a fluorescent donor (Cy3) and on its 5'-end with a fluorescentacceptor (Cy5).Formation of the fully wrapped sructure was accompanied by extensive fluorescence resonance energy transfer (FRET)from CY3 to Cy5 since the two ends of (dT)_(66) are in close prximity in the fully wrapped complex.Our results indicate that initial ssDNA binding to the tetramer is very rapid,with a biomolecular rate constant,k_1 app,of nearly 10~9M~(-1)s~(-1) in thelimit of low salt concentration,whereas the rate of dissociation is very low at all salt concentrationsthat were examined.However,the rate of initial binding and the rate of formation of the fully wrapped complex are identical.However,the rate of initial binding and the rate of formation of the fully wrapped complex are identical,indicating that the rate of wrapping of the ssDNA around the SSB tetramer is very rapid,with a lower limit rate of 700 s~(-1).The implications of this rapid bindingand wrapping reaction are discussed.