Functional Assessment of Population and Tumor-Associated APE1 Protein Variants

Abstract

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 APE1 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 APE1 missense mutations are seemingly rare and that the cancer-associated R237C variant may represent a reduced-function susceptibility allele.

Human Apurinic/Apyrimidinic Endonuclease 1

“…While studies are ongoing to identify a causal relationship between an APE1 defect and human disease, we provide in Table 2 a summary of the current list of APE1 missense variants reported to date, many of which are rare and have not been functionally characterized. Lastly, we note that re-sequencing of the APE1 exons in the 60 cancer cell lines within the NCI-60 cell line panel uncovered no novel non-synonymous mutations, suggesting that APE1 variants are not frequent (87).…”

“…and changes residue D148 to E148, did not alter the in vitro AP endonuclease efficiency of the encoded protein (69). D148E, as well as two other polymorphic variants (frequency of appearance > 3%), Q51H (rs1048945) and I64V (rs2307486), have since been shown to exhibit normal thermodynamic stability of protein folding, abasic endonuclease, 3¢-5¢ exonuclease and REF-1 activities, coordination during the early steps of BER in a simple reconstituted assay, and intra-cellular distribution when expressed exogenously in HeLa cells (87). A few missense variants, some of which were reported to be uniquely associated with amyotrophic lateral sclerosis (ALS), such as L104R, E126D, and D283G, were estimated or experimentally demonstrated to exhibit 40 to 90% reductions in AP endonuclease activity (69); however, as is discussed later, the validity of these variants is in question.…”

“…Two of the endometrial cancerassociated mutations resulted in an amino-acid substitution (P112L or R237C), with the other nucleotide change introducing a premature stop codon at residue W188 that would give rise to, minimally, a nuclease-inactive APE1 protein fragment. The P112L variant was found to exhibit normal activities in a range of biochemical and cell-based assays, whereas the R237C variant displayed reduced in vitro 3¢ to 5¢ exonuclease and 3¢-damage processing activities (87), which could have contributed to the genome instability and carcinogenic process by reducing overall proofreading capacity and strand-break repair. While studies are ongoing to identify a causal relationship between an APE1 defect and human disease, we provide in Table 2 a summary of the current list of APE1 missense variants reported to date, many of which are rare and have not been functionally characterized.…”

Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes

“…APE1 Proteins-Purified, recombinant, untagged human APE1 proteins used in this study were WT APE1, Q51H, I64V, P112L, D148E, R237C, G241R, P311S, and A317V described previously (26), and the APE1 mutants R237A (27) and Y128A (31). Four new mutants were created for assessment, i.e.…”

“…In the human population there are a number of naturally occurring variants of APE1, many of which have been assessed for AP endonuclease activity on DNA substrates to determine whether they show reduced activities potentially indicative of deleterious health effects (26,27). Although these variants have shown few differences in activity on short, naked DNA substrates, they have not been assessed for activities on a chromatin-relevant nucleosome, with which most DNA in the cell is associated.…”