Bacterial conjugation is a major route of horizontal gene transfer and a key driver of the dissemination of antibiotic resistance, exacerbating the global health crisis. Central to this process is TraI, a bifunctional relaxase-helicase encoded by the iconic F-plasmid family. TraI initiates single-stranded DNA (ssDNA) transfer by covalently attaching to plasmid DNA through its trans-esterase (TE) activity and subsequently unwinds the plasmid via its helicase activity. Although genetic and biochemical studies have postulated that two TraI molecules are required for efficient conjugative transfer, how these molecules coordinate their activities in space and time - and how strand nicking is coupled to helicase loading - has remained unknown. Here, we capture the elusive, functionally asymmetric TraI homodimer and report its cryo-EM structure, revealing that the interaction between the two TraI molecules is mediated predominantly by ssDNA. We further show that TE-driven duplex melting at the origin of transfer (oriT) by one TraI molecule enables helicase loading by a second TraI molecule on the transfer strand of the unnicked plasmid DNA. Although the TE domain is intrinsically competent for strand nicking, its activity is inhibited by the host factor IHF, and this inhibition is relieved upon helicase loading. Together, these findings define a regulatory loop that coordinates helicase loading with strand nicking, providing mechanistic insight into the earliest stages of conjugative DNA processing.