The bacteriophage T4 replication system is one of the best-studied of the related mechanims of DNA replication employed by prokaryotes and eukaryotes. The work described herein will focus on the DNA-dependent RNA polymerase encoded as gene product 61 and known as the primase enzyme. The primase contains a zinc ribbon motif which is currently emerging as an important feature of proteins involved in transactions of nucleic acids, including replication, transcription, and repair. The structure of the primase alone and in complexes will be interrogated by mutagenesis and metal-binding studies and ultimately by X-ray crystallography in collaboration with another laboratory. Its interaction with other proteins in the replication complex and with DNA will be studied by the determination of binding constants under various conditions, photocrosslinking experiments, by DNA footprinting studies, and by elucidation of the characteristics of and requirements for priming. Through these studies, the assembly and function of the primase/helicase primosome will be described in similar detail to that already determined for the polymerase holoenzyme.
