Structural basis of replication origin recognition by the DnaA protein.

ABSTRACTEscherichia coli DnaA binds to 9 bp sequences(DnaA boxes) in the replication origin, oriC, to forma complex initiating chromosomal DNA replication.In the present study, we determined the crystalstructure of its DNA-binding domain (domain IV)complexed with a DnaA box at 2.1 A˚ resolution.DnaA domain IV contains a helix–turn–helix motiffor DNA binding. One helix and a loop of the helix–turn–helix motif are inserted into the major grooveand 5 bp (3¢ two-thirds of the DnaA box sequence)are recognized through base-specific hydrogenbonds and van der Waals contacts with the C5-methyl groups of thymines. In the minor groove,Arg399, located in the loop adjacent to the motif,recognizes three more base pairs (5¢ one-third of theDnaA box sequence) by base-specific hydrogenbonds. DNA bending by ~28L¯was also observed inthe complex. These base-specific interactionsexplain how DnaA exhibits higher affinity for thestrong DnaA boxes (R1, R2 and R4) than the weakDnaA boxes (R3 and M) in the replication origin.INTRODUCTIONIn Escherichia coli, the oriC region (245 bp), where chromo-somal DNA replication is initiated (1), contains five distrib-uted binding sites (DnaA boxes) for the DnaA protein (2,3), akey factor for the initiation of chromosomal DNA replication.In the first step of replication initiation, DnaA binds to theDnaA boxes to form a multimeric nucleoprotein complexcontaining approximately 20 DnaA molecules at the oriCregion (initial complex formation) (1,4). The formation of thismultimeric protein–DNA complex is thought to be a commonstep in chromosomal replication for all cellular organisms(5,6). DnaA has high affinity for both ATP and ADP, and onlythe ATP-bound DnaA can unwind the double helix of theAT-rich 13mer sequence repeats, which are located near theDnaA-binding sites within oriC (open complex formation)(4,7,8). The DnaB helicase is assembled onto the opencomplex and forms a mobile complex with the DnaG primase(1). After primer RNA synthesis by DnaG, a homodimer of theb subunit of DNA polymerase III (pol III) is assembled on theoriC region, and promotes complementary strand synthesis.The pol III b subunit is a sliding clamp that binds the DNA andaccelerates the hydrolysis of DnaA-bound ATP in thepresence of the Hda/IdaB protein, which probably mediatesthe DnaA–b subunit interaction. This DnaA inactivationprevents extra initiations of replication within a single cellcycle (9,10).The DnaA protein is highly conserved among bacteria(11–13), and the eukaryotic counterpart is also found as asubunit of the origin recognition complex (5,14). Sequencealignments of the DnaA proteins have suggested that theprotein is composed of four domains, I, II, III and IV (15,16).Domain I is involved in the DnaA–DnaA interaction, whiledomain II does not seem to have a replication initiationfunction. Domain III contains the Walker-type ATPase motif,and is considered to be a major region for the DnaA–DnaAinteraction. Domain IV is the DNA-binding region, whichspecifically binds to the DnaA box sequence. Recently, thecrystal structure of the DNA-free DnaA protein domainsIII/IV from the thermophile Aquifex aeolicus was determined;however, the orientations of domains III and IV in thisstructure exhibited a steric clash with DNA, when the DNAwas modeled on the structure (17). A large conformationalchange of the DnaA domains III and IV must be required uponDNA binding.In order to initiate chromosomal DNA replication, DnaAspecifically recognizes 9 bp of the five DnaA boxes, R1, R2,R3, R4 and M, in the oriC region. The in vivo footprintinganalysis in synchronized cells revealed that DnaA binds to theR1, R2 and R4 sites throughout most of the cell cycle (18,19).Biochemical experiments have shown that DnaA binds to the