BIOPHYSICAL AND SCANNING FORCE MICROSCOPY STUDIES

该项目的主要目标是确定蛋白质-蛋白质和蛋白质-DNA相互作用在人类碱基切除-修复(BER)途径中的作用，并确定这些相互作用在不同的多态等位基因之间可能存在怎样的变化。BER是修复单碱基损伤的主要细胞机制。这种损害是由各种内源性和外源性因素引起的，这些因素导致碱基的氧化、烷基化或水解。例如，A和C残基通过碱基上的氨基的水解而自发脱氨；而电离辐射或博莱霉素等药物处理的氧化损伤导致碱性位点和链断裂，导致3‘末端受阻。这两种类型的损伤都会导致DNA中的碱性位点(脱氨的碱基被特定的DNA糖基酶去除)，并且都会被BER修复。人类的BER是由AP内切酶启动的，AP内切酶将5‘端的磷酸二酯键水解性地裂解到碱性位点，留下5’DRP位点和3‘OH。DNA聚合酶β随后将DNA链延长一个核苷酸，并去除具有DRP裂解酶活性的DRP位点，导致含有缺口的双链DNA。缺口随后被DNA连接酶(DNA连接酶I或DNA连接酶III，在修复蛋白XRCC1存在的情况下)封闭。BER涉及修复蛋白和DNA之间的一系列复杂的相互作用。AP内切酶、DNA Polβ和DNA连接酶I(或III)都能催化DNA上的反应。此外，体外实验表明，DNA polβ与DNA连接酶I和xrcc1结合，而xrcc1又与DNA连接酶III结合。这些观察表明了一个过程，其中结合事件的空间和时间序列控制着BER协同DNA修复的效率。要了解BER基因的遗传多态如何影响DNA修复，最重要的是了解它们相互作用的细节。为了进一步阐明BER的大分子相互作用的机制，我们准备投入大量的精力进行生物物理溶液状态分析以及BER系统组件的扫描力显微镜分析。这种分子水平的知识显然将直接在细胞水平上提供更多的理解，并对BER协同DNA修复的重要性有更好的看法。我们将解决的具体问题是：1)参与修复的其他蛋白质的存在如何影响蛋白质-DNA复合体的构象？2)蛋白质之间的结合亲和力是什么？它们是否受到损伤DNA的存在的影响。3)多态对上述属性有何影响？

The main goal of this project to determine the role of protein- protein and protein-DNA interactions in the human base excision- repair (BER) pathway, and to determine how these interactions may vary among polymorphic alleles. BER is the primary cellular mechanism by which single base lesions are repaired. Such lesions result from a variety of endogenous and exogenous agents that cause the oxidation, alkylation, or hydrolysis of bases. For example, A and C residues are spontaneously deaminated by the hydrolysis of the amino groups on the bases; while, oxidative damage by ionizing radiation or treatment with agents such as bleomycin leads to abasic sites and to strand scission, resulting in blocked 3'termini. Both types of damage result in abasic sites in the DNA (the deaminated bases are removed by specific DNA glycosylases) and both are repaired by BER. BER in humans is initiated by AP endonuclease which hydrolyticly cleaves the phosphodiester bond 5' to the abasic site, leaving a 5' dRP site and a 3'OH. DNA polymerase beta subsequently extends the DNA chain by one nucleotide and removes the dRP site with its dRP lyase activity, resulting in a double-stranded DNA containing a nick. The nick is subsequently sealed by a DNA ligase (either DNA ligase I or DNA ligase III in the presence of the repair protein XRCC1). BER involves a complex set of interactions between the repair proteins and DNA. AP endonuclease, DNA pol beta, and DNA ligase I (or III) all catalyze reactions on DNA. In addition, in vitro evidence indicates that DNA pol beta binds to DNA ligase I and to xrcc1, which binds to DNA ligase III. Finally, AP endonuclease and xrrc1 have been shown to affect the activity of DNA pol beta. These observations suggest a process wherein both a spatial and temporal sequence of binding events govern the efficiency of BER concerted DNA repair. To understand how genetic polymorphisms in BER genes might affect DNA repair, it is paramount to understand the details of their interactions. To further elucidate the mechanisms involved in the macromolecular interplay of BER, we are prepared to invest considerable effort towards the biophysical solution-state analyses, as well as scanning force microscopy analyses of the components of the BER system. Such knowledge at this molecular level will obviously impart directly a much greater understanding at the cellular level and a greater perspective of the importance of BER concerted DNA repair. The specific questions that we will address are: 1) How are the conformations of the protein-DNA complexes affected by the presence of the other proteins involved in the repair? 2) What are the binding affinities of the proteins for one another and are they affected by the presence of damaged DNA. 3) How do polymorphisms affect the above properties?