A very important feature of replication initiation proteins is the ability to bind to DNA via a characteristic domain. Usually, one or two DNA-binding domains can be distinguished within each initiator. In this work, we specified a new family of replication initiation proteins (the TrfA-like protein family) with unique domain compositions that are important for interactions with DNA. Using phylogenetic analysis and structure prediction methods simultaneously with biochemical assays, we demonstrate that in the replication initiator of the broad-host-range plasmid RK2, in addition to two winged helix domains, a third domain that interacts with DNA can be described. Mass spectrometric analysis followed by site-directed mutagenesis and in vitro and in vivo analysis of TrfA variants showed that DNA binding by all three domains of TrfA is important for stable nucleoprotein complex formation and the replication activity of the initiator.