GENE AMPLIFICATION BY A TRANSPOSITION APPARATUS

Georgetown University, Washington DC

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Abstract

Initiation of DNA replication coupled to recombination is a critical process for genetic exchange and DNA repair as well as for chromosomal replication. For proper maintenance of their chromosome, cells must finely regulate recombination and DNA replication linked to DNA exchange, carrying out processes such as double strand break repair while keeping the generation of genetic diversity to a tolerable level. The mechanism by which bacteriophage Mu DNA replication. For Mu transposition the phage- encoded transposase (MuA) assembles into a transpososome at the Mu ends, promoting transfer of the Mu ends to target DNA and forming a potential Mu replication fork at each end. The transposome than undergoes a transition to a replisome in a reaction specifically requiring the phiX174- type primosome, an apparatus that has been implicated in host DNA replication coupled to homologous recombination. The transition ascertains that specific host replication proteins such as the DnaB helicase and the DNA polymerase III holoenzyme are engaged at the Mu replication form while blocking access of the form to other host enzymes such as helicase II and DNA polymerase I. All proteins needed to initiate Mu DNA replication have been identified except for proteins present in a host enzyme fraction called Mu Replication Factor (MRF) alpha2, which acts together with molecular chaperone ClpX to disassemble the transpososome and assemble a pre-replisome. This investigation is focused on identify host proteins that make up MRFalpha2 and characterizing how the specific host proteins are sequentially assembled at the Mu replication fork. The hypothesis is that the Mu transposition system is designed to exploit the host apparatus for linking DNA replication to homologous recombination, and the long-term goal is to understand how the host proteins involved in Mu replication may function in cellular DNA replication linked to recombination. The specific aims are: 1) Identify host factor(s) characterize the nucleoprotein complexes at the potential Mu replication forks during transition from transposome to replisome; 3) examine what constitutes the assembly site of the phiX-type primosome at the Mu fork, determining the DNA structure and protein functions needed to promote primosome assembly.