Stopped-flow kinetic analysis of replication protein A-binding DNA: damage recognition and affinity for single-stranded DNA reveal differential contributions of k(on) and k(off) 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 greatest 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.
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
S M
SM
Patrick
J J
JJ
Turchi
D001483:Base Sequence; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D051716:Replication Protein A
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051716:Replication Protein A; D002945:Cisplatin