GEORGETOWN UNIVERSITY (THE)
This project examines the cellular mechanisms that promote initiation of DNA synthesis li DNA recombination. Such processes playa vital role in genetic exchange, double-strand break repair, and restart of replication forks. Bacteriophage Mu replicates its DNA through the process of transposition, and host factors (Mu Replication Factors or MRF) promote the seamless transition from phage-encoded transpososome to host-encoded replisome that catalyzes Mu DNA replication. The transpososome promotes strand exchange to create Mu DNA forks and then remains tightly bound to these forks. This nucleoprotein complex at the Mu fork (strand transfer complex or STC) is progressively remodeled by disassembly of the transpososome and sequential assembly of new nucleoprotein complexes that lead to replisome assembly. One group of MRFs are the restart proteins, which include Escherichia coli PriA, PriB, PriC, and DnaT, and they function to load the major replicative helicase DnaB on the Mu fork. Additional MRFs assembled on the Mu fork upstream of the restart proteins can influence the pathway by which DnaB loading takes place. One of these factors has been identified to be a truncated version of the translation initiation factor 2 (IF2), which promotes initiation of DNA replication by one of the two major restart mechanisms, the PriA-PriC pathway. A major hypothesis is that the MRFs playa crucial role at the interface between host recombination and replication functions to support and regulate initiation of DNA synthesis by the restart pathways. The thinking is that defining the functions required for Mu replication will unravel the complex design of the restart machinery and its versatility in promoting and regulating replisome assembly on various DNA templates. Pathways for Mu replication and restart will be examined by: 1) examining the role of the translation Initiation Factor 2 (IF2) in the PriA-PriC pathway for Mu DNA replication and host restart and 2) examining the function of the PriA, PriC, and DnaT proteins in these processes. These studies should further define the function of transition mechanisms that link recombination with the assembly of the replisome by restart functions. In human cells the enzymatic apparatus that functions at the interface between recombination and replication may play varied roles such as tumor suppression and prevention of premature aging. This work will further define the cellular apparatus that maintains the stability and integrity of the genome, preventing genetic alterations associated with cellular dysfunction.