Pre-steady-state binding of damaged DNA by XPC-hHR23B reveals a kinetic mechanism for damage discrimination.
The XPC-hHR23B complex (XPC-hHR23B) is a heterodimeric protein required for the initial step of DNA damage recognition in the global nucleotide excision repair (NER) pathway. A strong preference of XPC-hHR23B for UV- and cisplatin-damaged DNA has previously been demonstrated using equilibrium binding assays. To better understand the molecular mechanism of damage recognition by XPC-hHR23B, we carried out the pre-steady-state kinetic analysis of the XPC-hHR23B-DNA interactions using a stopped-flow fluorescence assay. XPC-hHR23B displays a faster k(on) for cisplatin- and UV-damaged duplex DNA than for undamaged DNA, with additional, minor effects on the k(off) rates. XPC-hHR23B has a high affinity for undamaged single-stranded DNA compared to duplex DNA, which can be largely attributed to a high rate of association. However, cisplatin damage on single-stranded DNA reduced the overall level of binding by a factor of 7, with nearly equal contributions from changes to the k(on) and k(off) rates. Together, these results support a model for initial damage recognition by XPC-hHR23B that is dependent on structural changes in the DNA, and not adduct chemistry.
Trego
Kelly S
KS
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Turchi
John J
JJ
D001483:Base Sequence; D001665:Binding Sites; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D007700:Kinetics; D008969:Molecular Sequence Data; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; C513287:RAD23B protein, human; C086985:RAD23A protein, human; C086984:XPC protein, human; D004247:DNA; D045643:DNA Repair Enzymes; D002945:Cisplatin