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ORC formation is required for proper bacterial origin function
Author(s) -
Miller Diana T,
Grimwade Julia,
Torgue Julien,
Leonard Alan
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a658-b
Subject(s) - dnaa , origin of replication , origin recognition complex , pre replication complex , microbiology and biotechnology , biology , dna replication , helicase , seqa protein domain , dna , genetics , chemistry , gene , eukaryotic dna replication , rna
Initiation of chromosome replication is precisely timed during the cell cycle so that all daughter cells receive equivalent genomes. In E. coli , regulation of initiation is focused on assembly of complexes comprising the chromosomal replication origin oriC and the initiator protein DnaA. DnaA is structurally homologous to CDC6/ORC1 proteins and forms an origin recognition complex (ORC), binding to high affinity oriC sites during most of the cell cycle. ORC transitions to a pre‐replication complex (pre‐RC) by additional DnaA binding to weaker affinity sites, causing DNA strand separation and helicase recruitment. Although the position and strength of DnaA binding sites are similar among all Enterbacteria, it is not clear what aspects of this conserved arrangement are required for ORC to correctly switch to pre‐RC. To address this, a series of DnaA binding mutants in oriC were constructed. The functionality of the mutants relative to wild‐type oriC was examined, and ORC and pre‐RC formation were analyzed by in vivo and in vitro dimethyl‐sulfate footprinting. We found that there is considerable plasticity in ORC assembly; as long as two strong DnaA sites are maintained in each half of oriC , ORC can form and transition to a pre‐RC. However, if only one strong site remains, oriC function is compromised. These data indicate that formation of ORC is a necessary first stage in replication initiation. Supported by NIH GM54042.