Thursday, April 3, 2025
4/3/2025
Coordination of DNA Metabolism by Replication Protein A - Summary DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. The precise mechanisms of how RPA imparts functional specificity is poorly resolved. Towards addressing this gap in knowledge, our long-term goals are to answer the following questions: a) RPA physically interacts with over three dozen DNA processing enzymes. How are these interactions determined, regulated, and prioritized? b) RPA binds to ssDNA with high affinity (KD <10-10 M). How do DNA metabolic enzymes that bind to ssDNA with hundred-fold lower affinities remove RPA? c) RPA plays a role in positioning the recruited enzymes (with appropriate polarity) onto the DNA. What are the structural, kinetic, and thermodynamic properties that regulate this process? d) How are the DNA and protein interaction activities of RPA tuned by post translational modifications such as phosphorylation? RPA achieves functional dexterity through a multi-domained architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. This dynamic plasticity has hindered structural, biochemical, and biophysical investigations of full-length RPA. Over the past eight years, our group has developed non-canonical amino acid based site-specific fluorescence labeling tools to investigate the dynamics of the individual domains of RPA. While difficult to accomplish, our breakthrough enabled us to reestablish how the individual domains of RPA bound, dissociated, and remodeled during various DNA metabolic processes. The findings were in stark contrast to commonly assumed models for RPA function and has opened numerous avenues to finally investigate and establish how RPA functions in specific DNA metabolic processes. For example, we showed that the commonly assumed high-affinity DNA binding domains of RPA were in fact the most dynamic and not bound to ssDNA in the context of the full-length protein. Utilizing our powerful biochemical, structural, and biophysical toolkit we here seek to resolve how RPA functions in the context of nucleosomes, R-loops, telomere, and in other DNA repair pathways.
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