A Molecular Mechanism Allowing Transposon Tn7 To Target Active Dna Replication
Transposons are jumping genes that are ubiquitous and abundant in all domains of life. They can move between locations that lack homology within a genome. Transposons drive the evolution of genomes through gene inactivation, expression modulation, and genome rearrangement. The bacterial transposon Tn7 and its relatives are widespread in diverse bacteria, likely due to their ability to control the frequency and targeting of transposition. My work presented here focuses on understanding the molecular mechanism of the TnsABC+E pathway of Tn7 transposition that preferentially targets actively transferring mobile DNA. I was able to establish a sensitive in vitro system for this transposition reaction with purified proteins and gapped DNA substrates preloaded with the [beta]-clamp. The transposition profile recapitulates that observed in vivo, indicating that the minimal features recognized by TnsE to target DNA replication are 3' recessed ends found in target DNA and the [beta]-clamp processivity factor (DnaN). I further show that the TnsE-[beta] interaction is largely conserved among Tn7-like elements; however, this interaction is also species specific. In a heterologous expression study, I found that TnsE homologs from Idiomarina loihiensis and Shewanella baltica only promoted transposition when DnaN from the same host was used in the cell. I propose that TnsE may have evolved to interact with the more variable portion of the clamp to avoid interfering with the normal traffic on the clamp. In an effort to screen for host proteins that may affect TnsE-mediated transposition, I found and confirmed an interaction between TnsE and SeqA, a protein involved in replication initiation control and organizing newly replicated chromosome DNA. Results from genetic studies support a model where TnsE interacts with SeqA to disrupt the SeqA superstructure that tracks with the replication fork. I also show that in wild type background, TnsE is able to direct transposition into the origin region and DNA undergoing leading-strand replication, consistent with the emerging picture that both the leading-strand and lagging-strand DNA replication are essentially discontinuous. These data point to a perspective of using Tn7 as a genetic tool in understanding the replication and repair processes in the cell.
Peters, Joseph E.
Helmann, John D; Roberts, Jeffrey Warren
Ph.D. of Microbiology
Doctor of Philosophy
Dissertation or Thesis