PNNL

Abstract

Nucleotide excision repair (NER) is a crucial pathway in the maintenance of genome stability requiring at least two dozen proteins. XPA and RPA have essential roles in the damage recognition step of NER. To better understand the mechanism of their interactions with DNA, we utilized equilibrium and stop-flow kinetic approaches with fluorescently labeled oligonucleotides. A circular plasmid with a single defined fluorescein was constructed to demonstrate that fluorescein is a bona fide NER lesion repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in subsequent studies. Oligonucleotide fluorescence was quenched upon specific binding to full-length recombinant Xenopus XPA (xXPA) and/or human RPA. The binding was highly sensitive to the buffer conditions. Analysis of equilibrium binding data with ds DNA and xXPA revealed a single dissociation constant (Kd) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant kon of 9.03 x108 M-1s-1 and koff of 26.1 s-1. From the ratio of off-rate to on-rate a calculated Kd of 28.9 nM was obtained, revealing that the kinetic and equilibrium studies were consistent. The affinity of xXPA for ds undamaged DNA determined in our spectrofluorimetry experiments was up to three orders of magnitude higher than previously reported values using different substrates, conditions, and assays [gel-shifts (EMSA), filter-binding, anisotropy, and surface plasmon resonance]. The same substrate DNA containing a 4-bp mismatch in the middle yielded a Kd five times higher (158 nM), indicating weaker binding by xXPA. This result is consistent with a model that XPA more readily dissociates from DNA containing single-stranded regions, perhaps to allow binding by other NER proteins. Only minor fluorescence changes upon interaction of xXPA with ss 50 mer were observed.