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 p rotein-DNA interactions. Multiple protein-binding sites are often invo lved. For example, the replication of plasmid R6K involves binding of the initiator protein pi to seven 22-bp direct repeats (DR) in the gam ma origin of replication (gamma osi). A mutant protein pi S87N has bee n isolated, that in Tris borate buffer (TB) binds cooperatively to sev en DR, whereas wild-type (wt) pi binds independently [Filutowicz et al ., Nucleic Acids Res. 22 (1994) 4211-4215]. Surprisingly, we found tha t wt pi can also bind cooperatively when Tris acetate (TA), Tris succi nate or Tris glutamate buffers are used instead of TB. The cooperative binding of the wt pi protein was also observed in the TB buffer at hi gh concentrations of Na(2)EDTA. These results suggest that pi may be a ble to assume two functionally distinct conformations as a result of e ither mutation or buffer composition. Moreover, we found that the mode of pi binding is determined not by the composition of the buffer in w hich the reaction was assembled, but by the composition of the electro phoresis buffer. We discuss the general implications of these findings .