SINGLE STRANDED DNA RECOGNITION IN TELOMERES

端粒是真核细胞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G的双链DNA片段组成，末端是3‘-单链突起。端粒特异的蛋白质与双链区和单链区结合形成一种独特的结构，其功能是保护染色体不受降解和端到端融合的影响，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、人类细胞系永生化和人类衰老中的作用，端粒复制和端粒长度的调节目前正在进行深入的研究。CDC13P是一种单链端粒DNA结合蛋白，从发芽酵母中鉴定出来。它是一种保护端粒末端免受降解的重要蛋白质，同时也是端粒酶活性的正负调节因子。在这项研究中，将使用生化和生物物理方法详细探讨酵母蛋白CDC13P的单链端粒DNA识别活性。到目前为止，几乎没有研究过具有序列特异性的结合单链DNA的蛋白质，也几乎不知道支配识别的分子相互作用。我们的建议的目的是：(1)生化探测单链端粒DNA的亲和力和CDC13 DNA结合域的特异性；(2)使用交联和突变来定位蛋白质/DNA相互作用的位置，并利用这些知识设计突变体用于活体研究(3)使用高分辨率异核核磁共振谱确定游离蛋白质及其DNA复合体的溶液结构。了解识别单链DNA的分子基础不仅在端粒复制和调控领域具有重要意义，而且对于涉及单链DNA的许多其他细胞过程，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.