The transposition of bacteriophage Mu is a means of both establishing lysogeny and replicating the genome during lytic development. The Mu transposition proteins, encoded by genes A and B, direct the 100 to 200- fold amplification of the phage genome in less than 40 min. These proteins promote transfer of Mu ends to target DNA, forming a branched intermediate. Replication of Mu DNA catalyzed by host replication proteins completes transposition. The Mu A protein is the component that catalyzes strand transfer, performing a function similar to retroviral integrases and other transposases. However, the participation of the Mu A protein is not over after catalyzing strand transfer. Tetrameric Mu A protein remains very tightly bound to the Mu ends, holding them together in a synaptic complex. The tightly bound Mu A protein blocks initiation of Mu DNA synthesis in a system of 8 purified Escherichia coli replication proteins, which can catalyze Mu DNA replication on the deproteinized strand transfer product. An additional, yet unidentified host factor allows initiation of Mu DNA synthesis on the native strand transfer product in a process that specifically requires E. coli DnaB helicase, DnaC protein, and the DNA pol III holoenzyme. The postulated function of the Mu A protein is to mediate the assembly of replication proteins at the Mu fork. The unidentified host factor may act as a Mu A-releasing factor, which may render the fork accessible to host proteins or participate in releasing the Mu A replication block once the replisome is assembled. The long-term goal is to understand how the Mu transposition apparatus is designed to promote high rates of Mu DNA amplification. The reasoning is that the Mu transposition apparatus most likely serves additional functions beyond the catalysis of strand transfer to assure high rates of Mu DNA synthesis. The objective of this project is to understand the function of Mu A protein and host factors in the initiation of Mu DNA synthesis and how the mechanisms involved are exploited in vivo to achieve high rates of Mu DNA synthesis. The specific objectives are to: 1) purify the Mu A-releasing factor needed to catalyze Mu DNA synthesis; 2) characterize assembly of E. coli replication proteins and disassembly of Mu A protein at the fork in vitro for the initiation of DNA synthesis; and 3) characterize how Mu DNA replicates and accumulates late in development when Mu DNA synthesis is proceeding at maximal rates. In the last specific objective, experimentation will focus on multiple initiation events late in Mu development and how successive rounds of Mu DNA synthesis are catalyzed. The knowledge gained from these analyses will help define how transposition proteins successfully exploits host enzymes for the amplification of the transposing element.
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