Protein Architecture and Remodeling in DNA Transposition and DNA Protection

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DESCRIPTION (provided by applicant): Higher-order protein-DNA complexes both orchestrate and catalyze many of life's most central processes. Genetic recombination and DMA protection, processes essential for maintaining the integrity of the genome for future generations, are especially rich in their use of protein-DNA superstructures. A key feature of these complexes is that they are often exceedingly stable, such that ATP-dependent protein-unfolding enzymes and proteases may be required to remodel, dismantle, destroy or recycle the component proteins. The focus of this project is to understand how protein complexes are recognized for remodeling or destruction by proteins of the Clp/Hsp100 ATPase family. Clp/Hsp100 proteins are a subfamily of the AAA+ enzymes that use ATP-hydrolysis to perform mechanical work on their substrates. The first specific goal is to understand how the protein-unfolding enzyme CIpX recognizes the protein-DNA complex that promotes DNA transposition of phage Mu. Experiments to elucidate the peptide signals within the transposase responsible for recognition are proposed. Furthermore, we will test a model in which asymmetric features of the complex guide the unfolding activity of CIpX to one specific transposase subunit, and thereby generate a new --less stable--complex with a unique architecture. The second goal is to elucidate how the DNA-protection protein Dps is recognized by CIpX and how this recognition is tuned to changing environmental conditions. The role of peptide signals, adaptor proteins and DNA in recognition of Dps will be investigated. Finally, proteomic experiments designed to achieve a global view of the role of ATP-dependent protein unfoldases/proteases are proposed. These experiments will give a proteome-wide view of the roles of specific adaptor proteins and peptide binding domains in substrate choice by AAA+ enzymes. Cellular mechanisms that protect the genome from damage, and promote the faithful repair of damaged DNA are critical to health and survival, as dramatically demonstrated by the numerous cancer syndromes associated with the genetic disruption of these cellular processes. Protein remodeling and destruction by AAA+ enzymes is a critical yet poorly understood aspect of the strategies used by cells to both interpret and protect their genomes.