Valinluck Victoria, Liu Pingfang, Burdzy Artur, Ryu Junichi, Sowers Lawrence C
Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, California 92350, USA.
Chem Res Toxicol. 2002 Dec;15(12):1595-601. doi: 10.1021/tx020062y.
To maintain genomic integrity, DNA repair enzymes continually remove damaged bases and lesions resulting from endogenous and exogenous processes. These repair enzymes must distinguish damaged bases from normal bases to prevent the inadvertent removal of normal bases, which would promote genomic instability. The mechanisms by which this high level of specificity is accomplished are as yet unresolved. One member of the uracil-DNA glycosylase family of repair enzymes, Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug), is reported to distinguish U:G mispairs from U:A base pairs based upon specific contacts with the mispaired guanine after flipping the target uracil out of the duplex. However, recent studies suggest other mechanisms for base selection, including local duplex stability. In this study, we used the modified base N6-methyladenine to probe the effect of local helix perturbation on Mug recognition of uracil. N6-Methyladenine is found in E. coli as part of both the mismatch repair and restriction-modification systems. In its cis isomer, N6-methyladenine destabilizes hydrogen bonding by interfering with pseudo-Watson-Crick base pairing. It is observed that the selection of uracil by Mug is sequence dependent and that uracil residues in sequences of reduced thermostability are preferentially removed. The replacement of adenine by N6-methyladenine increases the frequency of removal of the uracil residue paired opposite the modified adenine. These results are in accord with suggestions that local helix stability is an important determinant of base recognition by some DNA repair enzymes and provide a potential strategy for identifying the sequence location of modified bases in DNA.
为维持基因组完整性，DNA修复酶持续清除内源性和外源性过程产生的受损碱基和损伤。这些修复酶必须区分受损碱基和正常碱基，以防止意外去除正常碱基，否则会促进基因组不稳定。实现这种高度特异性的机制尚未得到解决。据报道，修复酶尿嘧啶-DNA糖基化酶家族的一个成员，大肠杆菌错配特异性尿嘧啶-DNA糖基化酶（Mug），在将目标尿嘧啶从双链体中翻转出来后，基于与错配鸟嘌呤的特定接触，区分U:G错配和U:A碱基对。然而，最近的研究提出了其他碱基选择机制，包括局部双链体稳定性。在本研究中，我们使用修饰碱基N6-甲基腺嘌呤来探究局部螺旋扰动对Mug识别尿嘧啶的影响。N6-甲基腺嘌呤在大肠杆菌中作为错配修复和限制修饰系统的一部分被发现。在其顺式异构体中，N6-甲基腺嘌呤通过干扰假沃森-克里克碱基配对破坏氢键。据观察，Mug对尿嘧啶的选择是序列依赖性的，并且热稳定性降低序列中的尿嘧啶残基被优先去除。用N6-甲基腺嘌呤取代腺嘌呤会增加与修饰腺嘌呤配对的尿嘧啶残基的去除频率。这些结果与局部螺旋稳定性是一些DNA修复酶碱基识别的重要决定因素的观点一致，并为识别DNA中修饰碱基的序列位置提供了一种潜在策略。
