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Mutational analysis reveals Escherichia coli oriC interacts with both DnaA‐ATP and DnaA‐ADP during pre‐RC assembly
Author(s) -
Grimwade Julia E.,
Torgue Julien J.C.,
McGarry Kevin C.,
Rozgaja Tanya,
Enloe Sareena T.,
Leonard Alan C.
Publication year - 2007
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2007.05930.x
Subject(s) - dnaa , biology , mutant , escherichia coli , plasmid , dnab helicase , dna , mutation , function (biology) , microbiology and biotechnology , biophysics , biochemistry , origin of replication , helicase , rna , gene
Summary 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.