We have determined the crystal structure of a monomeric biologically active form of the initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-Å resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal ␣4 and the N-terminal ␣4 helices, that recognize the 5 half and the 3 half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in ␣4 , whereas the ␣4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely ␣2 and ␣5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.initiator structure ͉ replication control ͉ replication initiation I n a major class of drug resistance plasmids, interaction of a plasmid-encoded replication initiator protein with the originproximal, cognate, repeated sequences called iterons, initiates Cairns-type replication that generates -shaped replication intermediates (1-12). Although a wealth of information exists on the genetics and biochemistry of replication initiation and its control in a few model plasmid systems, there has been a paucity of information on the atomic structure of the plasmid-encoded replication initiator proteins and their detailed structurefunction analysis. In fact, the structure of only one plasmidencoded initiator, namely RepE of F factor, is known to date (13). Unfortunately, the F plasmid system offers very few mutants of the RepE initiator that have been isolated and analyzed to date and very little published information exists on the interaction of the RepE initiator with host-encoded replication proteins.By contrast, the R6K plasmid system has a rich repertoire of biological and biochemical information on its initiator protein called (2,3,6,8,(14)(15)(16)(17)(18)(19), including its interaction with several host-encoded replication proteins (20-22), and its self interaction, via dimerization (23, 24), dimer-dimer (or dimer-monomer) interactions, that are essential for DNA looping (22). The R6K replicons consist of three replication origins called ␣, , and ␥. is a transacting initiator protein that binds to seven tandem iterons at ␥ (Fig. 1). Dimerization of negatively controls copy number, as revealed by the fact that mutations that enhance copy number also tend to monomerize the protein upon iteron binding (22,23,25). In addition to the dimerization interphase, a second type ofcontact is required for dimer-dimer or, more likel...