Mutational analysis reveals Escherichia coli oriC interacts with both DnaA-ATP and DnaA-ADP during pre-RC assembly.
Prior to initiating DNA synthesis, Escherichia coli oriC switches from ORC, comprising initiator DnaA bound at three high-affinity sites, to pre-RC, when additional DnaA molecules interact with low-affinity sites. Two types of low-affinity sites exist: R boxes that bind DnaA-ATP and DnaA-ADP with equal affinity, and I-sites with a three- to fourfold preference for DnaA-ATP. To assess the regulatory role of weak DnaA interactions during pre-RC assembly in vivo, we compared the behaviour of plasmid-borne wild-type oriC with mutants having an increased or decreased number of DnaA-ATP discriminatory I-sites. Increasing the number of discriminatory sites by replacing R5M with I2 inactivated extrachromosomal oriC function. Mutants with no discriminatory sites perturbed host growth and rapidly replaced wild-type chromosomal oriC, but normal function returned if one I-site was restored at either the I2, I3 or R5M position. These observations are consistent with assembly of E. coli pre-RC in vivo from mixtures of DnaA-ATP and DnaA-ADP, with I-site interactions coupling pre-RC assembly to DnaA-ATP levels.
Grimwade
Julia E
JE
Department of Biological Sciences, Florida Institute of Technology, 150 W. University Blvd., Melbourne, FL 32901, USA.
Torgue
Julien J-C
JJ
McGarry
Kevin C
KC
Rozgaja
Tanya
T
Enloe
Sareena T
ST
Leonard
Alan C
AC
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004252:DNA Mutational Analysis; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate