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镁对碱基切除修复酶至关重要,它会抑制N-甲基嘌呤-DNA糖基化酶的底物结合。

Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase.

作者信息

Adhikari Sanjay, Toretsky Jeffery A, Yuan Linshan, Roy Rabindra

机构信息

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.

出版信息

J Biol Chem. 2006 Oct 6;281(40):29525-32. doi: 10.1074/jbc.M602673200. Epub 2006 Aug 9.

Abstract

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. MPG activity and other DNA glycosylases do not have an absolute requirement for a cofactor. In contrast, all downstream activities of major base excision repair proteins, such as apurinic/apyrimidinic endonuclease, DNA polymerase beta, and ligases, require Mg(2+). Here we have demonstrated that Mg(2+) can be significantly inhibitory toward MPG activity depending on its concentration but independent of substrate type. The pre-steady-state kinetics suggests that Mg(2+) at high but physiologic concentrations decreases the amount of active enzyme concentrations. Steady-state inhibition kinetics showed that Mg(2+) affected K(m), but not V(max), and the inhibition could be reversed by EDTA but not by DNA. At low concentration, Mg(2+) stimulated the enzyme activity only with hypoxanthine but not ethenoadenine. Real-time binding experiments using surface plasmon resonance spectroscopy showed that the pronounced inhibition of activity was due to inhibition in substrate binding. Nonetheless, the glycosidic bond cleavage step was not affected. These results altogether suggest that Mg(2+) inhibits MPG activity by abrogating substrate binding. Because Mg(2+) is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.

摘要

N-甲基嘌呤-DNA糖基化酶(MPG)通过去除多种烷基化、脱氨基和脂质过氧化诱导的嘌呤加合物,启动DNA中的碱基切除修复。MPG活性和其他DNA糖基化酶对辅因子没有绝对需求。相比之下,主要碱基切除修复蛋白的所有下游活性,如脱嘌呤/脱嘧啶内切核酸酶、DNA聚合酶β和连接酶,都需要Mg(2+)。在此我们证明,Mg(2+)根据其浓度可对MPG活性产生显著抑制作用,但与底物类型无关。稳态前动力学表明,高浓度但生理浓度的Mg(2+)会降低活性酶浓度的量。稳态抑制动力学表明,Mg(2+)影响Km,但不影响Vmax,且这种抑制作用可被EDTA逆转,而不能被DNA逆转。在低浓度下,Mg(2+)仅对次黄嘌呤刺激酶活性,而对乙烯腺嘌呤无此作用。使用表面等离子体共振光谱的实时结合实验表明,活性的显著抑制是由于底物结合受到抑制。尽管如此,糖苷键裂解步骤不受影响。这些结果共同表明,Mg(2+)通过废除底物结合来抑制MPG活性。由于Mg(2+)是主要碱基切除修复酶下游活性的绝对需求,它可能作为碱基切除修复途径的调节剂,以有效且平衡地修复受损碱基,这些受损碱基通常比其后续修复产物中间体毒性和/或致突变性更低。

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