SINGLE STRANDED DNA RECOGNITION IN TELOMERES

端粒是真核生物染色体末端的脱氧核糖核蛋白结构。 它们由以3 '-单链突出端结束的双链体富含G的DNA的重复片段组成。 端粒特异性蛋白结合双链和单链区域以形成独特的结构，其功能是保护染色体免于降解和端对端融合，介导染色体分离，并通过充当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处于紧张的研究中。Cdc 13 p是一种单链端粒DNA结合蛋白，从芽殖酵母酿酒酵母中遗传鉴定。 它是一种保护端粒末端不被降解的必需蛋白质，并作为端粒酶活性的正性和负性调节剂。 在这项研究中，单链端粒DNA识别活性的酵母蛋白Cdc 13 p将详细探讨使用生物化学和生物物理方法。 到目前为止，很少有蛋白质被研究，结合单链DNA的序列特异性，并很少有人知道的分子相互作用，管理识别。 我们的目标是：（1）生物化学探针单链端粒DNA的亲和力和特异性的Cdc 13 DNA结合域;（2）使用交联和诱变定位的网站，蛋白质/DNA相互作用，并使用这些知识的突变体的设计在体内研究;（3）确定的自由蛋白质和它的DNA复合物的溶液结构，使用高分辨率的异频NMR光谱。理解单链DNA识别的分子基础不仅在端粒复制和调控领域，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.