C. Margulies et Jm. Kaguni, ORDERED AND SEQUENTIAL BINDING OF DNAA PROTEIN TO ORIC, THE CHROMOSOMAL ORIGIN OF ESCHERICHIA-COLI, The Journal of biological chemistry, 271(29), 1996, pp. 17035-17040
DnaA protein of Escherichia coil acts in initiation of chromosomal DNA
replication by binding specific sequences, termed DnaA boxes in the c
hromosomal origin, oriC, On binding, it induces a localized unwinding
to create a structure recognized by other replication proteins that ac
t subsequently in the initiation process, In this report, we examined
the binding of DnaA protein to each of the DnaA boxes in oriC, By gel
mobility shift assays, DnaA protein formed at least six discrete compl
exes. ATP or ADP included in the reaction mixture prior to electrophor
esis was required, Chemical cleavage of isolated complexes with 1,10-p
henanthroline-copper revealed that DnaA protein binds in an ordered ma
nner to the DnaA boxes in oriC, Preferential binding to one DnaA box (
RA) was confirmed by demonstration that a DNA fragment containing it w
as bound with greater affinity than another DnaA box sequence (R1), In
vitro replication activity correlated with a complex formed at a rati
o of 30 DnaA monomers/oriC in which all DnaA boxes are occupied, The l
ast site bound is DnaA box R3. This event may be critical in promoting
initiation from oriC as it correlates with in vivo observations that
binding of DnaA protein to box R3 occurs at the time of initiation of
chromosomal replication, whereas other DnaA boxes are bound by DnaA pr
otein throughout the cell cycle (Cassler, M, R,, Grimwade, J. E,, and
Leonard, A, C, (1995) EMBO J, 14, 5833-5841).