Hedglin Mark, O'Brien Patrick J
Chemical Biology Program, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
Biochemistry. 2008 Nov 4;47(44):11434-45. doi: 10.1021/bi801046y. Epub 2008 Oct 8.
DNA repair proteins conduct a genome-wide search to detect and repair sites of DNA damage wherever they occur. Human alkyladenine DNA glycosylase (AAG) is responsible for recognizing a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the base excision repair pathway. We have investigated the mechanism by which AAG locates sites of damage using an oligonucleotide substrate containing two sites of DNA damage. This substrate was designed so that AAG randomly binds to either of the two lesions. AAG-catalyzed base excision creates a repair intermediate, and the subsequent partitioning between dissociation and diffusion to the second site can be quantified from the rates of formation of the different products. Our results demonstrate that AAG has the ability to slide for short distances along DNA at physiological salt concentrations. The processivity of AAG decreases with increasing ionic strength to become fully distributive at high ionic strengths, suggesting that electrostatic interactions between the negatively charged DNA and the positively charged DNA binding surface are important for nonspecific DNA binding. Although the amino terminus of the protein is dispensable for glycosylase activity at a single site, we find that deletion of the 80 amino-terminal amino acids significantly decreases the processivity of AAG. These observations support the idea that diffusion on undamaged DNA contributes to the search for sites of DNA damage.
DNA修复蛋白会在全基因组范围内进行搜索,以检测并修复任何位置出现的DNA损伤位点。人类烷基腺嘌呤DNA糖基化酶(AAG)负责识别多种碱基损伤,包括烷基化和脱氨基的嘌呤,并通过碱基切除修复途径启动对它们的修复。我们利用一种含有两个DNA损伤位点的寡核苷酸底物,研究了AAG定位损伤位点的机制。设计该底物的目的是使AAG随机结合到两个损伤位点中的任意一个。AAG催化的碱基切除会产生一个修复中间体,随后解离与扩散到第二个位点之间的分配情况可根据不同产物的形成速率来量化。我们的结果表明,在生理盐浓度下,AAG有能力沿着DNA短距离滑动。随着离子强度增加,AAG的持续性降低,在高离子强度下完全变为非持续性,这表明带负电荷的DNA与带正电荷的DNA结合表面之间的静电相互作用对于非特异性DNA结合很重要。虽然该蛋白的氨基末端对于单个位点的糖基化酶活性并非必需,但我们发现删除80个氨基末端氨基酸会显著降低AAG的持续性。这些观察结果支持了这样一种观点,即在未损伤的DNA上扩散有助于寻找DNA损伤位点。