Nucleic Acids Research, Vol 27, Issue 23 4570-4576, Copyright © 1999 by Oxford University Press
MR Cassler, JE Grimwade, KC McGarry, RT Mott and AC Leonard
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.
ARTICLES
Drunken-cell footprints: nuclease treatment of ethanol-permeabilized bacteria reveals an initiation-like nucleoprotein complex in stationary phase replication origins
Department of Biological Sciences, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL 32901, USA.
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