Marc Wold
University of Iowa, USA
Title: DNA repair depends on specialized DNA-binding by replication protein A
Biography
Biography: Marc Wold
Abstract
Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. RPA is a highly flexible complex composed of three subunits, RPA1, RPA2 and RPA3 that contain six DNA binding domains (DBDs). One of the recurring mysteries of RPA is that the affinity of RPA for ssDNA does not directly correlate with cellular function. There are RPA mutations that reduce the affinity of the complex by two orders of magnitude are fully active in vitro and in cells, while other mutations that display higher affinity for ssDNA are partially or completely inactive. In particular, we have characterized a set of separation-of-function mutants in RPA1 that have mutations in pairs of aromatic residues in the DNA binding sites of DBD-A or -B. These mutants support DNA replication but are defective in multiple DNA repair pathways including NER and double strand break repair. These results suggest that replication and repair require different RPA-DNA interactions
We utilized single molecule total internal fluorescence microscopy (smTIRF) and ensemble assays to determine the molecular basis of this separation of function phenotype. We analyzed the kinetics of binding to different DNA structures including single-stranded intermediates found at sites of damage and replication. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions, and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA-DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions.