Analysis of the role of origin recognition complexes in the control of initiation of DNA replication in Escherichia coli

Chromosome duplication requires precisely timed formation of initiator protein complexes at replication origins that separate the DNA helix to allow DNA polymerase to begin synthesizing new strands. In Escherichia coli, this complex is composed of initiator protein DnaA bound to specific 9 base pair sites in the unique origin of chromosome replication oriC. Affinity of DnaA for its binding sites in oriC is determined by slight dissimilarities in nucleotide sequence. The highest affinity sites R1, R2, and R4 are persistently bound throughout the cell cycle and lower affinity sites bind coincident with initiation. In eukaryotes, origins are designated by complexes of initiator proteins (ORC) that recruit additional proteins and transition to pre-replication complexes (pre-RCs).;Based upon temporal similarities, we proposed that the persistent of complex of DnaA bound to its high affinity sites functions in a manner homologous to the eukaryotic ORC. To determine if the prokaryotic complex is also functionally similar to its eukaryotic homologue, mutational studies and dimethyl sulfate (DMS) footprinting were performed. Cooperativity among high affinity DnaA boxes was shown to play a critical role in facilitating the binding of DnaA to weak boxes in oriC. Weakening the three high affinity sites in oriC eradicated origin function in vivo and weak sites could not bind DnaA in vitro. Origins with a sole high affinity site were also nonfunctional but exhibited DnaA binding at proximal weak sites in vitro. Functionality was restored when two high affinity sites were present, provided that R1 was intact and the two high affinity sites were located in opposite halves of the origin. Weak sites in wild type oriC remained unbound by oligomerization-deficient DnaA, indicating that initiator protein interactions are required for the bacterial ORC to transition to a pre-RC. Data presented here indicate that the complex of DnaA bound to its high affinity sites in oriC is functionally analogous to the eukaryotic ORC and suggest that similar mechanisms control the initiation of DNA synthesis across all domains of life.