Buffer composition mediates a switch between cooperative and independent binding of an initiator protein to DNA

The regulation of many biological processes, including DNA replication, is frequently achieved by protein-protein interactions, as well as protein-DNA interactions. Multiple protein-binding sites are often involved. For example, the replication of plasmid R6K involves binding of the initiator protein n to seven 22-bp direct repeats (DR) in the γ origin of replication (γ ori). A mutant protein πS87N has been isolated, that in Tris·borate buffer (TB) binds cooperatively to seven DR, whereas wild-type (wt) π binds independently [Filutowicz et al., Nucleic Acids Res. 22 (1994) 4211–4215]. Surprisingly, we found that wt π can also bind cooperatively when Tris·acetate (TA), Tris·succinate or Tris·glutamate buffers are used instead of TB. The cooperative binding of the wt π protein was also observed in the TB buffer at high concentrations of Na 2EDTA. These results suggest that n may be able to assume two functionally distinct conformations as a result of either mutation or buffer composition. Moreover, we found that the mode of n binding is determined not by the composition of the buffer in which the reaction was assembled, but by the composition of the electrophoresis buffer. We discuss the general implications of these findings.