Effects of Backbone Contacts 3' to the Abasic Site on the Cleavage and the Product Binding by Human Apurinic/Apyrimidinic Endonuclease (APE1)
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- 作者：Tadahide Izumi ; Catherine H. Schein ; Numan Oezguen ; Yanling Feng ; and Werner Braun
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：January 27, 2004
- 年：2004
- 卷：43
- 期：3
- 页码：684 - 689
- 全文大小：109K
- 年卷期：v.43,no.3(January 27, 2004)
- ISSN：1520-4995
文摘
The mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) is a multifunctional proteinthat plays essential roles in DNA repair and gene regulation. We decomposed the APEs into 12 blocks ofhighly conserved sequence and structure (molegos). This analysis suggested that residues in molegoscommon to all APEs, but not to the less specific nuclease, DNase I, would dictate enhanced binding todamaged DNA. To test this hypothesis, alanine was substituted for N226 and N229, which form hydrogenbonds to the DNA backbone 3' of the AP sites in crystal structures of the APE1/DNA complex. Whilethe cleavage rate at AP sites of both N226A and N229A mutants increased, their ability to bind to damagedDNA decreased. The ability of a double mutant (N226A/N229A) to bind damaged DNA was furtherdecreased, while the Vmax was almost identical to that of the wild-type APE1. A double mutant at N226and R177, a residue that binds to the same phosphate as N229, had a significantly decreased activity andsubstrate binding. As the affinity for product DNA was decreased in all the mutants, the enhanced reactionrate of the single mutants could be due to alleviation of product inhibition of the enzyme. We concludethat hydrogen bonds to phosphate groups 3' to the cleavage site is essential for APE1's binding to theproduct DNA, which may be necessary for efficient functioning of the base excision repair pathway. Theresults indicate that the molego analysis can aid in the redesign of proteins with altered binding affinityand activity.