Chepanoske Cindy Lou, Lukianova Olga A, Lombard Murielle, Golinelli-Cohen Marie-Pierre, David Sheila S
Department of Chemistry, University of Utah, 315 S 1400 E, Salt Lake City, Utah 84112-0850, USA.
Biochemistry. 2004 Jan 27;43(3):651-62. doi: 10.1021/bi035537e.
MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate.
MutY是碱基切除修复（BER）超家族中的一种腺嘌呤糖基化酶，参与DNA中7,8 - 二氢 - 8 - 氧代 - 2'-脱氧鸟苷（OG）:A和G:A错配的修复。MutY包含一个[4Fe - 4S]2+簇，它是一种新型DNA结合基序的一部分，称为铁硫簇环（FCL）基序。该基序存在于BER糖基化酶超家族的一部分成员中，定义了核酸内切酶III样亚家族。位点特异性交联成功用于研究MutY的DNA - 蛋白质界面。与OG:A错配相邻掺入的光反应性核苷酸4 - 硫代胸腺嘧啶（4ST）在底物DNA和MutY之间形成了特异性交联。通过正离子电喷雾电离（ESI）串联质谱对参与交联反应的氨基酸进行了表征。该分析揭示了MutY中Arg 143为修饰位点。Arg 143和附近的Arg 147在整个核酸内切酶III样亚家族中是保守的。通过定点诱变将Arg 143和Arg 147替换为丙氨酸会降低MutY对OG:A和G:A错配的腺嘌呤糖基化酶活性。此外，R143A和R147A酶对含有与OG和G相对的底物类似物2'-脱氧 - 2'-氟腺苷的双链体的亲和力降低。使用核酸内切酶III - DNA复合物结构对与DNA结合的MutY进行建模表明，这两个保守的精氨酸位于靠近DNA主链的位置。质谱实验的见解与功能诱变结果相结合表明，含[4Fe - 4S]2+簇亚家族中的这两个氨基酸在识别受损DNA底物中起重要作用。
