A Novel Action of Human Apurinic/Apyrimidinic Endonuclease

Abstract

␤-L-Dioxolane-cytidine (L-OddC, BCH-4556, Troxacitabine) is a novel unnatural stereochemical nucleoside analog that is under phase II clinical study for cancer treatment. This nucleoside analog could be phosphorylated and subsequently incorporated into the 3 terminus of DNA. The cytotoxicity of L-OddC was correlated with the amount of L-OddCMP in DNA, which depends on the incorporation by DNA polymerases and the removal by exonucleases. Here we reported the purification and identification of the major enzyme that could preferentially remove L-OddCMP compared with dCMP from the 3 termini of DNA in human cells. Surprisingly, this enzyme was found to be apurinic/apyrimidinic endonuclease (APE1) (1), a well characterized DNA base excision repair protein. APE1 preferred to remove L-over D-configuration nucleosides from 3 termini of DNA. The efficiency of removal of these deoxycytidine analogs were as follows:This report is the first demonstration that an exonuclease can preferentially excise L-configuration nucleoside analogs. This discovery suggests that APE1 could be critical for the activity of L-OddC or other L-nucleoside analogs and may play additional important roles in cells that were not previously known.Deoxyribonucleoside analogs are among the most effective agents for the treatment of cancer and viral diseases. Most of these compounds exert their function by inhibiting cellular or viral DNA synthesis. Since the naturally occurring nucleosides are in the ␤-D-configuration, most of the nucleoside analogs were designed in that configuration. The discovery of ␤-L-2Ј,3Ј-dideoxy-3Ј-thiocytidine (L-SddC, 3TC), 1 a stereochemically unnatural L-nucleoside analog (Fig. 1) inhibitor of HIV and hepatitis B virus replication (2-6), defined a new category for the design of nucleoside analogs. Among these, ␤-L-dioxolane-cytidine (L-OddC) (Fig. 1) is the first to show potential anticancer activity (2, 3), and clinical evaluation demonstrates its effectiveness against both leukemia and solid tumors (4 -7). For activation, most nucleoside analogs require conversion to the triphosphate metabolite by several cellular enzymes. Our previous studies have shown that L-OddC can be phosphorylated by deoxycytidine kinase to its monophosphate metabolite, which is further phosphorylated by cellular kinases to its di-and triphosphate metabolites (2, 3). The latter form of L-OddC can be incorporated into DNA by DNA polymerases ␣, ␤, ␦, ␥, and ⑀ in vitro (8). Since L-OddC lacks a hydroxyl group at the 3Ј-position, it causes premature termination of DNA replication once incorporated and eventually leads to cell death. This chain termination is probably the major mechanism of action of L-OddC. We have also shown previously that the cytotoxicity of this drug is directly related to the steady-state level of L-OddC in DNA (2).The steady-state level of L-OddC in DNA is not only dependent on incorporation by DNA polymerases but also on the excision by DNA repair enzymes. We have previously observed that incorporated L-OddCMP could be excised fr...

Human Apurinic/Apyrimidinic Endonuclease (Ape1) and Its N-terminal Truncated Form (AN34) Are Involved in DNA Fragmentation during Apoptosis

“…The 3Ј-5Ј-exonuclease activity of normal Ape1 is 2-4 orders of magnitude less than the AP endonuclease activity of Ape1 (19,20). However, Chou et al (32,33) recently demonstrated that Ape1 possesses a specialized 3Ј-5Ј-exonuclease activity that preferentially removes mismatched deoxyribonucleotides at the 3Ј termini of nicked DNA. We find that AN34/Ape1 purified from apoptotic cells possesses enhanced exonuclease activity, which hydrolyzes the 3Ј-DNA terminus from normal deoxyribonucleotides more efficiently than does normal Ape1.…”

Structure and Expression of the TREX1 and TREX2 3′→5′ Exonuclease Genes

“…Conserved residues in these enzymes indicate a common catalytic mechanism involving a single metal ion. The 3Ј excision activity of the APEX protein is relatively weak and appears to be influenced by substrate and reaction conditions (37) as well as by the structure of the 3Ј terminal nucleotide (38,39). The structure of the p53 protein is similar to the exonuclease III and APEX proteins (40), and p53 protein is reported to contain 3Ј 3 5Ј exonuclease activity (7).…”

Modulation of the 3′→5′-Exonuclease Activity of Human Apurinic Endonuclease (Ape1) by Its 5′-incised Abasic DNA Product

“…In contrast, the robust AP endonuclease activity of Ape1 is related to the demonstrated roles in BER of exonuclease III in Escherichia coli or Apn1 in yeast (1,13). Recently, it was shown that Ape1 acting as an exonuclease can remove therapeutic antitumor nucleoside analogs incorporated at the 3Ј ends of DNA oligonucleotides, which suggested that Ape1 contributes to cellular resistance to these drugs (20). This work was further extended to show that the exonuclease activity of Ape1 is enhanced on DNA mispairs at the 3Ј termini of nicks and gaps (21).…”