Binding studies of human single-stranded DNA binding and repair proteins: Replication protein A and RAD52

Heterotrimeric replication protein A (RPA) is the primary eukaryotic single-stranded DNA (ssDNA) binding protein that plays an essential role in DNA metabolism including replication, recombination and repair. RPA has 6 ssDNA binding domains (DBDs) A-F. It prefers poly-pyrimidine over poly-purine sequences and binds non-canonical DNA structures (G-quadruplexes and triplexes). In order to study the crystal structure of RPA bound to ssDNA, SELEX experiments were performed to identify a specific ssDNA binding sequence for RPA. SELEX experiments with the primary DBDs-A and -B select an 8 nucleotide TC-rich motif, whereas DBDs-C, -D and -E retrieve a 20 nucleotide G-quadruplex forming sequence. Fluorescence polarization binding studies with RPA-AB and RPA-CDE-core show that these DBDs bind pyrimidine- and purine-rich mixed sequences similarly. Interestingly, RPA-DE binds preferentially to the G-quadruplex sequence, a unique preference not observed with other RPA constructs studied. Circular dichroism experiments show RPA-CDE unfolding the G-quadruplex while RPA-DE stabilizes it. Binding of RPA70-C, a construct of RPA that has not been previously purified, to various DNA sequences indicates a universal binder function for this domain. Circular dichroism titration experiments with a longer G-quadruplex forming sequence, containing a TC-rich region 5' to the G-quadruplex, is not unfolded by RPA-CDE-core possibly because RPA70-C binds the TC-rich region while RPA-DE binds the G-quadruplex. Molecular modeling studies of RPA32-D and proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA32-D and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties.;RAD52 and RPA are two key players in the repair of double-stranded DNA breaks (DSBs). The exact mechanism of recruitment of these proteins to damaged DNA is unknown. A small section of this thesis, dedicated to studying the ssDNA binding properties of phosphorylated RPA (pRPA) and RAD52, indicated that pRPA binds with a greater affinity to a mixed ssDNA sequence when compared to RPA, and RAD52 binds ssDNA ends. These results combined with previous studies from the Borgstahl laboratory suggest that phosphorylation of RPA may serve to recruit downstream repair proteins like RAD52 in order to efficiently repair damaged DNA.