SINGLE STRANDED DNA RECOGNITION IN TELOMERES

端粒是在真核生物染色体末端发现的脱氧核糖核蛋白结构。它们由重复的富含双g的DNA束组成，以3'-单链悬垂结尾。端粒特有的蛋白质结合双链和单链区域形成独特的结构，其功能是保护染色体免受降解和端到端融合，介导染色体分离，并通过作为端粒酶的底物来调节细胞的复制潜力。由于端粒在肿瘤发生、人类细胞系的永生和人类衰老中发挥着重要作用，端粒复制和端粒长度的调控目前正受到广泛的研究。Cdc13p是一种单链端粒dna结合蛋白，从酿酒酵母中遗传鉴定出来。它是一种重要的蛋白质，保护端粒的末端免受降解，并作为端粒酶活性的积极和消极调节者。本研究将采用生物化学和生物物理方法对酵母蛋白Cdc13p的单链端粒DNA识别活性进行详细探讨。迄今为止，很少有蛋白质被研究以序列特异性结合单链DNA，并且对控制识别的分子相互作用知之甚少。我们的建议的目的是：(1)生物化学探针单链端粒DNA的亲和力和特异性的Cdc13 DNA结合域；(2)使用交联和诱变来定位蛋白质/DNA相互作用的位点，并将这些知识用于设计体内研究的突变体。(3)使用高分辨率异核磁共振波谱确定游离蛋白质及其DNA复合物的溶液结构。了解单链DNA识别的分子基础不仅在端粒复制和调控领域具有重要意义，而且对许多其他涉及单链DNA的细胞过程，如转录、复制、修复和同源重组都具有重要意义。

Telomeres are the deoxyribonucleoprotein structures found at the ends of eukaryotic chromosomes. They are composed of repetitive tracts of duplex G-rich DNA ending in a 3'-single-stranded overhang. Proteins specific to telomeres bind both the double and single-stranded regions to form a distinct structure that functions to protect chromosomes from degradation and end-to-end fusion, to mediate chromosome segregation, and the modulate the replicative potential of the cell by acting as substrates for the enzyme telomerase. Telomeric replication and the regulation of telomere length are currently under intense study because of the role telomeres play in tumorigenesis, the immortalization of human cell lines, and human aging. Cdc13p is a single-stranded telomeric DNA-binding protein identified genetically from the budding yeast Saccharomyces cerevisiae. It is an essential protein which protects the ends of telomeres from degradation and acts as both a positive and negative regulator of telomerase activity. In this study, the single-stranded telomeric DNA recognition activity of the yeast protein Cdc13p will be probed in detail using both biochemical and biophysical approaches. To date, few proteins have been studied that bind single-stranded DNA with sequence specificity, and little is known about the molecular interactions that govern recognition. The aims of our proposal are to: (1) Biochemically probe single-stranded telomeric DNA affinity and specificity of the Cdc13 DNA-binding domain; (2) Use crosslinking and mutagenesis to localize the sites of protein/DNA interaction and use this knowledge for the design of mutants for in vivo studies (3) Determine the solution structures of free protein and its DNA complex using high-resolution heteronuclear NMR spectroscopy. Understanding the molecular basis of the recognition of single- stranded DNA has important implications not only in the field of telomere replication and regulation, but for many other cellular processes involving single-stranded DNA such as transcription, replication, repair, and homologous recombination.