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如何在多态性等位基因之间变化。BER是单基损伤修复的主要细胞机制。这种病变是由多种内源性和外源性物质引起的，这些物质会导致碱基的氧化、烷基化或水解。例如，A和C残基通过碱基上的氨基水解而自发脱氨；而电离辐射或博来霉素等药物的氧化损伤会导致碱基位点和链断裂，导致3'端受阻。这两种类型的损伤都导致DNA中的基本位点（脱氨基碱基被特定的DNA糖基酶去除），并且都被BER修复。人体内的BER是由AP内切酶引发的，AP内切酶水解磷酸二酯键5‘到碱性位点，留下一个5’ dRP位点和一个3'OH。DNA聚合酶β随后将DNA链延长一个核苷酸，并利用其dRP裂解酶活性去除dRP位点，从而产生含有缺口的双链DNA。缺口随后被DNA连接酶（在修复蛋白XRCC1的存在下，DNA连接酶I或DNA连接酶III）密封。BER涉及修复蛋白和DNA之间一系列复杂的相互作用。AP内切酶、DNA pol β和DNA连接酶I（或III）都能催化DNA上的反应。此外，体外证据表明DNA pol β与DNA连接酶I和xrcc1结合，xrcc1与DNA连接酶III结合。最后，AP内切酶和xrrc1已被证明影响DNA pol β的活性。这些观察结果表明，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基因的遗传多态性如何影响DNA修复，了解它们相互作用的细节是至关重要的。为了进一步阐明BER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，以及对BER系统组成部分的扫描力显微镜分析。这种分子水平上的知识显然会直接在细胞水平上给予更大的理解，并对BER协同DNA修复的重要性有更大的认识。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复合物的构象？2)蛋白质之间的结合亲和力是什么？它们是否受到受损DNA的影响？3)多态是如何影响上述特性的？

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?