Molecular Determinants of DNA Sliding and Hopping by a DNA Repair Glycosylase

描述(申请人提供)：人类基因组DNA中异常的尿嘧啶导致G：C到A：T颠换突变，这是在许多癌症中发现的最常见的突变。人类尿嘧啶DNA糖基酶(HAND)使用一种显着的搜索机制来搜索并移除脱氧核糖磷酸骨架上的尿嘧啶碱基，该机制使该酶能够在人类基因组中约60亿个正常碱基对的背景下快速定位罕见的病变部位。这种一般的搜索机制被称为“促进扩散”，蛋白质利用DNA链作为轨迹来加速其特定位置的定位。促进扩散的用途 两条微观传递路径：一维“滑动”和三维“跳跃”。虽然许多研究已经证实了这种普遍机制的存在，但“促进扩散”的细节仍然是个谜。这一建议的重点是阐明TH分子相互作用，这些分子相互作用对于HAN追踪DNA链是很重要的。我们最近开发的“分子时钟”(MC)方法利用一个小分子陷阱来捕获瞬时跳跃酶，而对滑动的跳跃酶的影响最小，从而允许单独解剖两条跟踪路径。使用这种方法可以探索的关键参数包括平均滑动距离的计算，酶与DNA的平均跳跃距离，以及DNA滑动的一维扩散常数。在第一个目标中，我们将使用MC方法来测试DNA跟踪的几个关键方面。首先，为了测试DNA磷酸盐静电学的作用，我们将在DNA骨架上由一定数量的碱基对分隔的两个尿嘧啶靶点之间插入中性甲基膦酸键，然后测量电荷消融对Hung在两个位点之间滑动的概率的影响。其次，我们将检验一个新的假设，即如果在两个尿嘧啶靶点之间插入热力学稳定的结合位点，则可以发生定向偏向转移(即使在没有提供能量的辅助因子的情况下)。这一目的将涉及在尿嘧啶位点之间插入间隔的高亲和力四氢呋喃基本SIE类似物。据预测，偏向行走将通过高亲和力的“岛屿转移”发生。使用碱性位点的偏向行走与体内情况高度相关，在体内情况下，Hung必须定位和切除聚集的尿嘧啶位点，例如在Ig体细胞超突变过程中。第二个目标是设计出具有更强追踪能力的悬肽尾部变体。利用表达蛋白连接技术(EPL)和/或化学连接技术构建HAND和短带正电的多肽之间的融合蛋白。这些多肽将来自几种DNA结合蛋白(P53、HOXD9、H3)，之所以选择它们，是因为它们代表不同的大小和总电荷，并已知影响DNA结合和结合动力学。我们将使用这些变体来测量基本的跟踪参数，这些参数将直接测试非特异性DNA上的停留时间和滑动的一维扩散常数如何影响体外和体内损伤修复的效率。

DESCRIPTION (provided by applicant): Aberrant uracils in human genomic DNA lead to G:C to A:T transversion mutation, the most frequent mutation found in many cancers. Human uracil DNA glycosylase (hUNG) searches and removes uracil bases from deoxyribose phosphate backbone using a remarkable search mechanism that allows the enzyme to rapidly locate rare lesion sites in a background of roughly 6 billion normal base pairs in the human genome. This general search mechanism has been referred to as "facilitated diffusion", where proteins utilize the DNA chain as a track to accelerate location of their specific sites. Facilitated diffusion uses two microscopic transfer pathways: one-dimensional "sliding" and three-dimensional "hopping". Although numerous studies have established the existence of such a general mechanism, the details of "facilitated diffusion" remain mysterious. The focus of this proposal is to elucidate th molecular interactions that are important for DNA chain tracking by hUNG. Our recently developed "molecular clock" (MC) approach utilizes a small molecule trap to capture transient hopping enzymes while having minimal effect on the sliding ones, thus allowing the dissection of the two tracking pathways individually. Key parameters that can be probed using this approach include the calculation of mean sliding distance, the average distance an enzyme hops away from DNA, and the 1D diffusion constant for DNA sliding. In the first aim, we will use MC approach to test several key aspects of DNA tracking. First, to test the role of DNA phosphate electrostatics, we will insert neutral methylphosphonate linkages in the DNA backbone between two uracil target sites separated by a set number of base pairs, and then measure the effect of charge ablation on the probability that hUNG will slide between the sites. Second, we will test a novel hypothesis that directionally-biased transfer can occur (even in the absence of an energy- providing cofactor) if thermodynamically stable binding sites are inserted between the two uracil target sites. This aim will involve the insertion of spaced high-affinity tetrahydrofuran abasic sie analogues between the uracil sites. The prediction is that a biased walk will occur via high-affinity "island transfer". A biased-walk using basic sites is highly relevant to the in vivo situaion where hUNG must locate and excise clustered uracil sites such as in the process of Ig somatic hypermutation. The second aim is to engineer hUNG-peptide tail variants to have enhanced tracking abilities. Fusion proteins between hUNG and short, positively charged peptides will be constructed using expressed protein ligation technology (EPL) and/or chemical ligation. These peptides will be derived from several DNA binding proteins (p53, HOXD9, H3), and are chosen because they represent various sizes and overall charge, and are known to affect DNA binding and association kinetics. We will measure the fundamental tracking parameters with these variants, which will directly test how the residence time on nonspecific DNA, and the 1D diffusion constant for sliding affect the efficiency of damage repair both in vitro and in vivo.