Stopped-flow kinetic analysis of replication protein A-binding DNA -: Damage recognition and affinity for single-stranded DNA reveal differential contributions of kon and koff rate constants

Replication protein A (RPA) is a heterotrimeric protein required for many DNA metabolic functions, including replication, recombination, and nucleotide excision repair (NER), We report the pre steady-state kinetic analysis of RPA-binding DNA substrates using a stopped-flow assay to elucidate the kinetics of DNA damage recognition. The bimolecular association rate, k(on), for RPA binding to duplex DNA substrates is great est for a 1,3d(GXG), intermediate for a 1,2d(GpG) cisplatin-DNA adduct, and least for an undamaged duplex DNA substrate. RPA displays a decreased k(on) and an increased k(off) for a single-stranded DNA substrate containing a single 1,2d(GpG) cisplatin-DNA adduct compared with an undamaged DNA substrate. The k(on) for RPA-binding single-stranded polypyrimidine sequences appears to be diffusion-limited. There is minimal difference in k(on) for varying length DNA substrates; therefore, the difference in equilibrium binding affinity is mainly attributed to the k(off). The k(on) for a purine rich 30-base DNA is reduced by a factor of 10 compared with a pyrimidine-rich DNA of identical length. These results provide insight into the mechanism of RPA-DNA binding and are consistent with RPA recognition of DNA-damage playing a critical role in NER.