DNA repair endonuclease ERCC1-XPF as a novel therapeutic target to overcome chemoresistance in cancer therapy

The ERCC1–XPF complex is a structure-specific endonuclease essential for the repair of DNA damage by the nucleotide excision repair pathway. It is also involved in other key cellular processes, including DNA interstrand crosslink (ICL) repair and DNA double-strand break (DSB) repair. New evidence has recently emerged, increasing our understanding of its requirement in these additional roles. In this review, we focus on the protein–protein and protein–DNA interactions made by the ERCC1 and XPF proteins and discuss how these coordinate ERCC1–XPF in its various roles. In a number of different cancers, high expression of ERCC1 has been linked to a poor response to platinum-based chemotherapy. We discuss prospects for the development of DNA repair inhibitors that target the activity, stability or protein interactions of the ERCC1–XPF complex as a novel therapeutic strategy to overcome chemoresistance

Functional Assessment of Population and Tumor-Associated APE1 Protein Variants

<div><p>Apurinic/apyrimidinic endonuclease 1 (APE1) is the predominant AP site repair enzyme in mammals. APE1 also maintains 3′–5′ exonuclease and 3′-repair activities, and regulates transcription factor DNA binding through its REF-1 function. Since complete or severe APE1 deficiency leads to embryonic lethality and cell death, it has been hypothesized that APE1 protein variants with slightly impaired function will contribute to disease etiology. Our data indicate that except for the endometrial cancer-associated APE1 variant R237C, the polymorphic variants Q51H, I64V and D148E, the rare population variants G241R, P311S and A317V, and the tumor-associated variant P112L exhibit normal thermodynamic stability of protein folding; abasic endonuclease, 3′–5′ exonuclease and REF-1 activities; coordination during the early steps of base excision repair; and intracellular distribution when expressed exogenously in HeLa cells. The R237C mutant displayed reduced AP-DNA complex stability, 3′–5′ exonuclease activity and 3′-damage processing. Re-sequencing of the exonic regions of <i>APE1</i> uncovered no novel amino acid substitutions in the 60 cancer cell lines of the NCI-60 panel, or in HeLa or T98G cancer cell lines; only the common D148E and Q51H variants were observed. Our results indicate that <i>APE1</i> missense mutations are seemingly rare and that the cancer-associated R237C variant may represent a reduced-function susceptibility allele.</p></div

Reconstitution assay using purified BER proteins.

<p>(<b>A</b>) Wild-type (WT) and variant APE1 proteins were incubated with UDG and POLβ with ³²P-labeled 34U DNA substrate (1 pmol), and the reactions were resolved on a urea-polyacrylamide denaturing sequencing gel. The non-incised substrate (S), AP site incision product (P1), and gap-filling extension product (P2) were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. (<b>B</b>) Relative AP site cleavage efficiency. Shown are the averages and standard deviations of 4 independent reactions. (<b>C</b>) Relative gap-filling activity. Shown are the average and standard deviation of 4 independent assays.</p

Variant proteins and thermodynamic stability of folding as determined by chemical denaturation.

<p>(<b>A</b>) Following purification, wild-type (WT) and variant APE1 proteins were quantified and analyzed (1 µg) by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Shown is a representative gel image. Molecular mass standards are indicated to the left in kDa. (<b>B</b>) Profile of maximum wavelength emission for tryptophan fluorescence relative to GdnHCl concentration for the wild-type (WT) and variant APE1 proteins (top). The free energies of protein unfolding in the absence of denaturant (ΔG_uw) and the values reflecting the dependence of the free energy on denaturant concentration (m_eq) are shown (bottom).</p

REF-1 assay.

<p>(<b>A</b>) HCT116 nuclear extract was incubated with ³²P-labeled consensus (CON) or mutant (MUT) AP-1 oligonucleotide substrates, and binding reactions were resolved on a non-denaturing polyacrylamide gel. Control reactions without nuclear extract (no extract) are shown. The arrow designates the position of the AP-1-specific consensus binding complex, not seen with the MUT double-stranded DNA. Higher molecular weight non-specific complexes are observed. (<b>B</b>) Reduced wild-type (WT) or variant APE1 protein was incubated with HCT116 nuclear extract in the presence of the ³²P-labeled AP-1 CON DNA substrate. Shown is the AP-1-specific complex in the absence (no protein) or presence of the indicated reduced APE1 protein after phosphorimager analysis. Plotted is the relative AP-1 DNA binding activity, in comparison with reduced WT protein. Values represent the average and standard deviation of 3 independent experimental points.</p

Intracellular localization of APE1 protein variants.

<p>(<b>A</b>) Representative microscopy images of the mCherry APE1 fusion proteins following plasmid transfection into HeLa cells. Shown are the DAPI nuclear staining, mCherry fusion protein fluorescence and the merged images. (<b>B</b>) Comparative cytoplasm to nuclear distribution for the different mCherry APE1 proteins. Using densitometry, the ratio of exogenous cytoplasmic mCherry-tagged wild-type (WT) APE1 protein to endogenous cytoplasmic protein was divided by the ratio of exogenous nuclear mCherry-tagged WT APE1 protein to endogenous nuclear protein, and this value was designated as 1. The identical ratio was then determined for each of the APE1 variant proteins, and plotted relative to the WT value. Shown is the average and standard deviation of results from 3 separate extract preparations and western blot experiments.</p

APE1 protein variants and oligonucleotide substrates.

<p>(<b>A</b>) Linear schematic of the 318 residue APE1 protein, including several reported amino acid substitutions. NLS = nuclear localization sequence; REF-1 = redox regulatory portion of the protein; italics = polymorphic variants; * = unique disease-associated variants; red text = variants with reduced AP endonuclease activity. The repair nuclease domain and several functionally important amino acids (C65, E96, D210, D283 and H309) are indicated. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065922#pone-0065922-t001" target="_blank">Table 1</a> for additional details. (<b>B</b>) The 18F NMR and 18G NMR oligonucleotides were used to design the double-stranded AP endonuclease substrate. (<b>C</b>) The 15P or 17P oligonucleotide was annealed to the 34G oligonucleotide to generate a 3′-recessed exonuclease/repair substrate, whereas the 34U oligonucleotide was annealed to 34G to create the uracil-containing duplex for the reconstitution assay. Oligonucleotides are written 5′ to 3′, with the non-labeled strands written upside-down. F = the AP site analog, tetrahydrofuran.</p

Re-sequencing of <i>APE1</i> exons in the NCI-60 cancer cell line panel.

<p>Re-sequencing was performed as described in Materials and Methods. All sequences were analyzed for homology to transcript variant 3 of APE1 (NM_080649.1). Genotypes have been divided into wild-type (D/D), heterozygous (D/E) or homozygous variant (E/E) for residue position 148 of APE1. Note: *, D/D for position 148, but Q/H heterozygous for position 51. Cancer type and cell line name are designated. Additional information is available at: <a href="http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html" target="_blank">http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html</a>.</p