This long running project has been focused on nucleoprotein complexes active in DNA recombination and replication. The main tools that we have been using are electron microscopy and computational image analysis. Over the course of this project new and innovative methods have been developed that greatly increase our ability to understand the structure and function of many different helical polymers. What emerges in our studies of proteins like bacterial RecA and human Rad51 is that the polymers they form are highly dynamic, and that these polymers cannot be reduced to a single structure. Rather, the multiplicity of states that can exist needs to be understood. The relevance of these studies to disease has been greatly elevated by the understanding that proteins such as the product of the breast cancer susceptibility gene BRCA2 interact with Rad51 and target Rad51 to sites of DNA damage. In the proposed extension of this grant there are four aims: I) continued studies of complexes between RecA/Rad51 and the proteins that regulate these filaments, such as DinI, BRCA2, Hed1 and RAD51AP1. A focus will be on how the C-terminal domain of RecA and the N-terminal domain of Rad51 are regulatory, and how regulatory proteins that modulate the properties of these filaments interact with these regulatory domains. II) Determination of the structure of the filaments formed by viral single-stranded DNA binding proteins. The initial focus will be on Herpes Simplex Type 1 Virus (HSV-1) ICP8, but will then be extended to related viral proteins, such as from Epstein Barr Virus and Kaposi Sarcoma Herpes Virus. III) Structural studies of F-pili, needed for bacterial conjugation, and filamentous bacteriophage. It has long been suspected that the two are structural homologs, but structural studies of these filaments have been very difficult. We now have the tools in place to determine fairly high resolution structures for both. F-pili are involved in the transfer of antibiotic resistance within a bacterial population, so the public health relevance is great. IV) Capsid proteins that form spherical viral shells can be mutated or modified so that they form helical tubes instead. These tubes contain a conformation of the protein that fails to switch properly, and are thus of great interest in understanding virus assembly. The proposed focus is on capsid proteins from bacteriophage P22, Infectious Bursal Disease Virus, and Murine Leukemia virus.
This project is aimed at understanding structure/function relationships in large complexes of proteins with DNA. Some of these proteins are active in genetic recombination, and defects in these recombination pathways can lead to cancer. Other complexes are involved in the transfer of antibiotic resistance within populations of bacteria, an area of growing concern for public health. And other complexes are involved in the assembly of viruses, including one that causes leukemia.
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