Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro.
BACKGROUND
Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo.


RESULTS
We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region.


CONCLUSIONS
SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.
Taghbalout
A
A
Institut Jacques Monod, CNRS, Université Paris 6-7, France.
Landoulsi
A
A
Kern
R
R
Yamazoe
M
M
Hiraga
S
S
Holland
B
B
Kohiyama
M
M
Malki
A
A
D020856:AT Rich Sequence / Q000502:physiology; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D051738:Origin Recognition Complex; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D018741:Replication Origin / Q000235:genetics*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000037:antagonists & inhibitors; D014157:Transcription Factors; D014764:Viral Proteins / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; C089746:SeqA protein, E coli; D014157:Transcription Factors; D014764:Viral Proteins; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499294:dam protein, E coli