Drunken-cell footprints: nuclease treatment of ethanol-permeabilized bacteria reveals an initiation-like nucleoprotein complex in stationary phase replication origins

Abstract

The nucleoprotein complex formed on oriC, the Escherichia coli replication origin, is dynamic. During the cell cycle, high levels of the initiator DnaA and a bending protein, IHF, bind to oriC at the time of initiation of DNA replication, while binding of Fis, another bending protein, is reduced. In order to probe the structure of nucleoprotein complexes at oriC in more detail, we have developed an in situ footprinting method, termed drunken-cell footprinting, that allows enzymatic DNA modifying reagents access to intracellular nucleoprotein complexes in E.coli, after a brief exposure to ethanol. With this method, we observed in situ binding of Fis to oriC in exponentially growing cells, and binding of IHF to oriC in stationary cells, using DNase I and Bst NI endonuclease, respectively. Increased binding of DnaA to oriC in stationary phase was also noted. Because binding of DnaA and IHF results in unwinding of oriC in vitro, P1 endonuclease was used to probe for intracellular unwinding of oriC. P1 cleavage sites, localized within the 13mer unwinding region of oriC ', were dramatically enhanced in stationary phase on wild-type origins, but not on mutant versions of oriC unable to unwind. These observations suggest that most oriC copies become unwound during stationary phase, forming an initiation-like nucleoprotein complex.