Wong Donny, DeMott Michael S, Demple Bruce
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
J Biol Chem. 2003 Sep 19;278(38):36242-9. doi: 10.1074/jbc.M306065200. Epub 2003 Jul 10.
The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta.
人类细胞中的主要无碱基内切核酸酶Ape1蛋白是一种多功能酶，在DNA碱基切除修复（BER）中起关键作用。除了作为BER中的脱嘌呤/脱嘧啶内切核酸酶的主要活性外，Ape1还具有对DNA内部切口和缺口的3'末端具有特异性的3'-磷酸二酯酶、3'-磷酸酶和3'→5'-核酸外切酶功能。核酸外切酶活性在3'错配处增强，这表明Ape1在BER中可能作为相对不准确的DNA聚合酶β的校对活性发挥作用。为了更精确地阐明这一作用，我们研究了Ape1降解模拟BER中间体的DNA底物的能力。我们发现Ape1核酸外切酶在错配和正确匹配的3'末端均有活性，且更倾向于错配。在我们的实验中，Ape1的核酸外切酶活性在单核苷酸缺口处比在DNA切口处更活跃，尽管后者应该是聚合酶β修复合成的产物。然而，核酸外切酶活性受到附近5'-切割的无碱基残基的抑制，这些残基是由Ape1的脱嘌呤/脱嘧啶内切核酸酶活性产生的。大肠杆菌内切核酸酶IV最近描述的核酸外切酶活性也是如此。Ape1的大肠杆菌同源物核酸外切酶III对不同底物没有区分。聚合酶β去除5'无碱基残基减轻了对Ape1核酸外切酶活性的抑制。这些结果表明，在DNA修复合成以及聚合酶β切除无碱基脱氧核糖-5-磷酸后，Ape1核酸外切酶在BER过程中发挥作用。
