Structural Biology of Multi-Domain Proteins and Multi-Protein Machinery in DNA Replication and Repair

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PROJECT SUMMARY Faithful replication of DNA and response to damaged DNA, essential to cell propagation and survival, proceeds via the action of multi-protein machines. While considerable progress has been made in elucidating the mechanisms of DNA replication and damage response from studies of bacteria and other lower organisms, information on humans is lacking because the protein sequences and structures are not conserved. Our long- term goals are to understand the action of human DNA replication and damage response machinery. Our strategy is to elucidate the structural basis for these processes using the full complement of modern structural biology tools and interpret this information using a range of biochemical and biological approaches. We have shown in the SV40 model system that the helicase that creates the single-stranded DNA (ssDNA) at the origin of replication also actively loads the human ssDNA binding protein replication protein A (RPA), and that this action is essential for DNA priming by DNA polymerase -primase (pol-prim). We have also characterized the complex and dynamic structural architectural of the modular RPA protein as it engages this ssDNA template and have begun to address the quandary of how RPA releases the template to hand off to pol-prim. Once the primase subunits are loaded on the DNA template, an ~10 nt RNA is synthesized and the primed template is transferred to the pol  subunits for extension to the final ~30 nt of RNA-DNA primer. Our goal is to continue to define the trajectory of actions on the template as it is replicated. The next phase of our research is to solve the fundamental mysteries about the release of RPA and loading of pol-prim onto the template, the counting of primer length by primase, and the hand-offs from primase to pol  and in turn to pol  or pol . Our DNA damage response research seeks to understand the mechanism of action of the key proteins involved, discern the biochemical bases for malfunctions caused by disease-associated mutations, and ultimately use this knowledge to help evaluate the potential of chemotherapies targeted to these proteins. Here again, RPA has a critical role, in this case, in recruiting partner proteins that stall and remodel replication forks and activate important signaling proteins. The goal is to obtain structural understanding and functional validation of the interactions between RPA and damage response proteins, and their role in responding to encounters with damaged DNA.