title: Equilibrium and stop-flow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein A.
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. Fluorescein is a bona fide NER lesion because a circular plasmid with a single defined fluorescein was repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in the 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 (K(d)) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant k(on) of 9.03 x 10(8) M(-1) s(-1) and k(off) of 26.1 s(-1). From the ratio of off-rate to on-rate, a calculated K(d) 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 spectrofluorometry experiments was up to 3 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 K(d) five times higher (158 nM), indicating weaker binding by xXPA. The differences in K(d) values for these two substrates were mainly attributable to the k(on), rather than k(off) rates. Fluorescence intensity changes upon interaction of xXPA with ss 50-mer were too low to calculate an accurate K(d). Although recombinant human RPA binding to the ds 50-mer was very weak (K(d) > 1 mM), stop-flow and equilibrium measurements to ss oligonucleotide yielded K(d) values of 96 and 20.3 nM, respectively, which correlated with previously reported values using gel mobility shift assays and a similarly sized poly-dT. Equilibrium and stop-flow measurements to the cognate and mismatched ds oligonucleotides using both xXPA and hRPA yielded a 2- to 3-fold increase in the K(d).
authors.affiliation: Pacific Northwest National Laboratory (PNNL), Molecular Biosciences Department, P.O. Box 999, Richland, Washington 99352, USA.
authors.forename: Lilia M
authors.initials: LM
authors.lastname: Iakoucheva
authors.forename: Randall K
authors.initials: RK
authors.lastname: Walker
authors.forename: Ben
authors.initials: B
authors.lastname: van Houten
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Ackerman
mesh_terms: D000818:Animals; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D051716:Replication Protein A; D013050:Spectrometry, Fluorescence; D014982:Xenopus laevis; D014983:Xeroderma Pigmentosum; D051760:Xeroderma Pigmentosum Group A Protein
chemical_list: D018736:DNA Adducts; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D016601:RNA-Binding Proteins; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein