Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Abstract

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPA^Cy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPA^Cy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

Keywords: DNA Binding Proteins; Fluorescence Enhancement through non canonical Amino Acids (FEncAA); Genetic code expansion (GCE); Nucleotide Excision Repair (NER); Protein Dynamics; Replication Protein A (RPA); Site-Specific Labeling; Xeroderma Pigmentosum group A (XPA).

Figure 1.. Position of 4AZP incorporation in XPA.

A) AlphaFold model of XPA (PDB: F2Z2T2) is shown with Arg-158 in the DNA binding domain highlighted. Arg-158 is replaced with 4AZP in XPA^4AZP and tethered to a fluorophore (Cy3) in XPA^Cy3. B) XPA from CryoEM structure of the TFIIH complex (6RO4) is shown with DNA depicted in black. The Zn²⁺ molecule is shown in grey. R158 is shown in red and is positioned close to the DNA, but does not make direct contact.

Figure 2.. Generation of XPA^Cy3 and characterization of DNA binding activity.

A) XPA, XPA^4AZP, and XPA^Cy3 were analyzed on SDS-PAGE and subjected to imaging using Coomassie staining (left) or fluorescence imaging (right). Only XPA^Cy3 is observed under the fluorescence channel. B) DNA binding activity of XPA, XPA^4AZP, and XPA^Cy3 were compared by monitoring the change in intrinsic Trp. fluorescence as a function of [DNA]. The DNA substrate has a 5′-(dT)₆₀ nt ssDNA overhang and a 13 bp dsDNA region. 100 nM protein was used in the experiment. Data were fit to a Menten-hyperbola to obtain apparent K_d values for binding. For XPA^Cy3, C) a quenching in Trp. fluorescence is observed upon binding to DNA. However, D) an enhancement in Cy3 fluorescence is observed in the presence of DNA. These data show that XPA^Cy3 does not show a loss in overall DNA binding activity and the position of the Cy3 produces an excellent change in fluorescence that can be used to monitor DNA binding dynamics.

Figure 3.. Assays to capture the DNA binding dynamics of XPA.

A) Stopped flow analysis of XPA^Cy3 binding to DNA was measured by rapidly mixing the protein and DNA (red) or buffer (black) and monitoring the change in Cy3 fluorescence. A rapid increase in Cy3 fluorescence is captured when XPA^Cy3 binds to DNA. The data is phenomenologically fit to a single exponential equation and yields k_obs = 0.12 s⁻¹. B) Single molecule total internal reflectance fluorescence (smTIRF) microscopy of DNA binding dynamics of XPA^Cy3. DNA was tethered to a glass surface. Cy5 on the DNA (as pictured) was used to identify spots of binding and then photobleached. Changes in Cy3 fluorescence were measured after flowing in XPA^Cy3. Three difference fluorescence states are observed likely denoting multiple configurational states of the bound XPA.

Figure 4.. Overproduction, purification, and labeling of XPA.

A) SDS-PAGE analysis of uninduced and IPTG induced cells and induction carried out at varying conditions as denoted. Ideal overproduction of XPA^4AZP is observed after overnight incubation at 16°C. SDS-PAGE analysis of protein samples from various fractionation steps during B) affinity purification on Ni²⁺-NTA and C) size exclusion chromatography. D) XPA^Cy3 samples before and after separation on Biogel-P4 are shown. E) Integrity of flash frozen XPA^Cy3 samples were assessed after multiple cycles of free-thaw cycles. XPA^Cy3 tolerates one freeze-thaw cycle when stored in 40% glycerol. F) XPA^Cy3 protein degrades when stored in buffer containing 10% glycerol. G) Fluorescence and H) Coomassie imaging of SDS-PAGE gels showing XPA^Cy3 stored in 40% glycerol. No degradation is observed when protein is left overnight at room temperature or at 4°C.