RECOGNITION AND REPAIR OF MISMATCHED DNA BY MUTY

由于越来越多的例子将功能失调的DNA修复与癌症联系起来，DNA修复已经引起了相当大的关注。特别是，鸟嘌呤的氧化损伤正在成为导致突变和致癌的重要致病事件。事实上，修复由7，8-二氢-8-氧-2‘-脱氧鸟苷(OG)引起的突变的复杂修复途径已经被发现。MutY通过从OG：A错配中去除错误结合的腺嘌呤残基，在防止突变方面发挥了重要作用。MutY属于碱基切除修复DNA糖基酶的超家族，包含a[4Fe-4S]簇，这在电子转移蛋白中更常见。因此，MutY不仅在维持高保真DNA复制方面具有重要的功能，而且还具有独特的结构和机械特性。该实验室开发了一个多方面的研究计划，旨在提供对MutY结构和功能特性的详细了解。我们的计划最初专注于研究MutY的基本性质，并澄清MutY催化的底物专一性和化学等重要问题。此外，我们还为MutY制备了新的不可切割底物类似物，它们是表征MutY-底物DNA复合体的有效底物模拟物。在对MutY的基本酶学性质有了充分的了解后，我们将把注意力转向更详细地研究MutY参与损伤的DNA识别和修复的性质。我们将研究四个特定的领域：(1)我们将进一步表征MutY识别和修复G：A和OG：A错配的内在化学。(2)我们将通过对错配进行特定的官能团替换或改变错配周围的序列来确定MutY所需的识别元件，以确定MutY识别和修复G：A和OG：A错配所涉及的内在性质。(3)我们将利用结构和生化方法研究MutY-DNA复合体的性质，以深入了解DNA修复酶对损伤特异性识别的相关因素。(4)通过对MutY[4Fe-4S]簇环基序氨基酸的特异性改变以及对MutY功能性质的影响，研究MutY[4Fe-4S]簇环基序在DNA识别中的作用。

DNA repair has attracted considerable attention due to increasing number of examples linking dysfunctional DNA repair with cancer. In particular, oxidative damage to guanine is emerging as an important causative event leading to mutagenesis and carcinogenesis. Indeed, complex repair pathways for the repair of mutations caused by 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) have been uncovered. MutY plays an important role in the prevention of mutations by removal of misincorporated adenine residues from OG:A mismatches. MutY belongs to a superfamily of base-excision repair DNA glycosylases and contains a a [4Fe-4S] cluster which is more commonly found in electron transfer proteins. Thus, MutY is not only functionally important in maintaining high fidelity DNA replication, it also has unique structural and mechanistic properties. This laboratory has developed a multifaceted research program aimed at providing a detailed understanding of the structural and functional properties of MutY. Our program has initially focused on investigating the basic properties of MutY and clarifying important issues of substrate specificity and chemistry catalyzed by MutY. In addition, we have prepared novel noncleavable substrate analogs for MutY that are effective substrate mimics to characteize the MutY-substrate DNA complex. With this solid understanding of the basic enzymatic properties of MutY, we will turn our attention to investigating in greater detail the properties of MutY involved in damaged DNA recognition and repair. Four specific areas will be examined: (1) We will further characterize the intrinsic chemistry of MutY's recognition and repair of G:A and OG:A mismatches. (2) We will determine the intrinsic properties of G:A and OG:A mismatches that are involved in their recognition and repair by MutY by making specific functional group substitutions on the mismatch or by altering the sequence surrounding the mismatch to determine the recognition elements required by MutY. (3) We will investigate the properties of the MutY-DNA complex using structural and biochemical methods to gain insight into factors involved in damage specific recognition by DNA repair enzymes. (4) We will investigate the role of MutY's [4Fe-4S] cluster loop motif (FCL) in damaged DNA recognition by making specific alterations in amino acids of the FCL and determining the effects on MutY's functional properties.