PHI29 DNA POLYMERASE AND TERMINAL PROTEIN

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DNA synthesis requires a preexisting primer for initiation, which is often in the form of an oligonucleotide. However, several viral-encoded DNA polymerases, such as hepatitis and human adenovirus, uniquely initiate replication from a ¿¿¿terminal protein¿¿¿ and prime elongation from a nucleotide. Phi29 bacteriophage of B. subtilis is the most biochemically well-characterized protein-primed replication system and it can therefore serve as a model for these more complex disease-causing viruses. Although there has been an extensive biochemical characterization of the initiation, transition, and elongation steps of Phi29 DNA replication, none of the steps have been structurally characterized at the atomic level. Therefore, we propose to use X-ray crystallography to visualize the three steps of Phi29DNA replication. We have already determined the structure of the apo polymerase and the polymerase complexed with single stranded DNA (manuscript in preparation). These structures have provided insight into the basis for the intramolecular strand displacement and processivity displayed by Phi29 DNA polymerase. We are currently determining the structure of the initiation complex (the polymerase with its priming protein, and/or template, and incoming nucleotide). We also plan to crystallize and determine the structures of the elongation and transition complexes. Analysis of these novel structures will allow us to understand protein priming in phi29. Such understanding will lend insight into the replication of related disease-causing viruses.