The Exonuclease Activity of Human Apurinic/Apyrimidinic Endonuclease (APE1)

Abstract

Human DNA apurinic/apyrimidinic endonuclease (APE1) plays a key role in the DNA base excision repair process. In this study, we further characterized the exonuclease activity of APE1. The magnesium requirement and pH dependence of the exonuclease and endonuclease activities of APE1 are significantly different. APE1 showed a similar K m value for matched, 3 mispaired, or nucleoside analog ␤-L-dioxolane-cytidine terminated nicked DNA as well as for DNA containing a tetrahydrofuran, an abasic site analog. The k cat for exonuclease activity on matched, 3 mispaired, and ␤-L-dioxolane-cytidine nicked DNA are 2.3, 61.2, and 98.8 min ؊1 , respectively, and 787.5 min ؊1 for APE1 endonuclease. Site-directed APE1 mutant proteins (E96A, E96Q, D210E, D210N, and H309N), which target amino acid residues in the endonuclease active site, also showed significant decrease in exonuclease activity. DNA base excision repair is the main pathway to repair DNA base damages caused by oxidation, radiation, and the loss of bases (1, 2). There are several enzymes that participate in this pathway, including DNA polymerase ␤, DNA ligase III-XRCC1 complex (1, 2), and apurinic/apyrimidinic endonuclease (APE1) 1 to protect the genome integrity (1-3). The DNA repair activity of APE1 resides in the C-terminal region (4, 5). It endonucleolytically makes a nick immediately adjacent to 5Ј of an apurinic/apyrimidinic (AP) site and generates a hydroxyl group at the 3Ј terminus upstream of the nick and a 5Ј-deoxyribose phosphate moiety downstream (6). DNA polymerase ␤ further processes the product of APE1 by inserting a nucleotide into the gap and releasing the 5Ј-deoxyribose phosphate by its intrinsic lyase activity (7). The repair process is then completed by either DNA ligase I or DNA ligase III/XRCC1 to seal the nicked DNA (1). During DNA base excision repair, DNA polymerase ␤ is the major polymerase that incorporates a nucleotide into the gapped DNA (1). However, DNA polymerase ␤ is a lower fidelity polymerase (8) in comparison to the replicating DNA polymerases ␦ or ⑀ (9). ⌻he error rate of polymerase ␤ is about 1 in 4000 incorporations (1).APE1 is a versatile multifunctional protein (10). In addition to its endonuclease activity, it also possesses 3Ј-phosphatase, 3Ј-phosphodiesterase, RNase H, and 3Ј-5Ј-exonuclease activities (10). APE1 knockout mice die in the early embryonic stage (11), which indicates this protein is critical for development. Previously we reported (12) that the 3Ј-5Ј-exonuclease activity of APE1 is the major exonuclease activity in the human cell nucleus for the removal of the nucleoside analog ␤-L-dioxolanecytidine (L-OddC, BCH-4556, Troxacitabine, and Troxatyl) from the 3Ј termini of DNA. L-OddC is a novel nucleoside analog with L-configuration that is currently under phase III clinical trial and is showing promising activity for the treatment of leukemia (13,14). The incorporation of L-OddC into DNA terminates DNA chain elongation because of the lack of the 3Ј-hydroxyl group on the sugar moiety of L-OddC. The cytotoxicity ...

Modulation of the 5′-Deoxyribose-5-phosphate Lyase and DNA Synthesis Activities of Mammalian DNA Polymerase β by Apurinic/Apyrimidinic Endonuclease 1

“…Magnesium is required for Ape1 enzymatic activity but also destabilizes the complex of Ape1 with the incised DNA product to facilitate turnover (38). The E96Q mutant form of Ape1 may be an interesting enzyme for subsequent analysis, because it has been reported to bind tightly to DNA containing tetrahydrofuran residues even in the presence of magnesium (51). However, pol ␤ undergoes significant conformational changes upon binding to divalent cations and during catalysis (52), so we cannot rule out the possibility that the absence of magnesium constrains pol ␤ in a conformation favorable for stable physical interactions with Ape1.…”

The 3′→5′ Exonuclease of Apn1 Provides an Alternative Pathway To Repair 7,8-Dihydro-8-Oxodeoxyguanosine in Saccharomyces cerevisiae

“…The standard reaction mixture (20 l) contained 3 nM 5Ј 32 P-labeled oligonucleotide duplex, 100 mM KCl, 20 mM HEPES/KOH, pH 7.6, 0.1 mg/ml bovine serum albumin (BSA), 1 mM dithiothreitol (DTT), 5 mM MgCl 2 and either 0.5 nM of Apn1 or 25 pM Xth, unless otherwise stated. For Ape1, the reaction buffer was 20 mM HEPES/KOH, pH 7.4, 0.1 mg/ml BSA, 30 mM KCl, 1 mM DTT, 1 mM MgCl 2 , and 1 nM enzyme (6 (25).…”

Human Apurinic/Apyrimidinic Endonuclease 1

“…The exonuclease activity of human APE1, however, is poorly processive and ‡ 100-fold less efficient than its AP endonuclease activity, although the former activity is somewhat influenced by reaction conditions, sequence/positional context (see comments in previous section), and thermal stability 682 LI AND WILSON of the duplex (28,44,45,226,231). Notably, APE1 displays the capacity to excise certain 3¢-mispaired nucleotides with increased proficiency, and, in this setting, may act as an autonomous proofreading enzyme for DNA polymerases which lack editing activity (28,226). Moreover, Chou et al (29) isolated APE1 as the major protein that excises the clinically relevant, chain-terminating nucleoside analog, b-L-dioxolanecytidine (also known as, troxacitabine), from the 3¢-end of a 3¢-recessed oligonucleotide substrate (see more in ''APE1 as a Therapeutic Target'').…”