Dynamic 31P NMR of Backbone Dynamics in DNA

DNA 骨架动力学的动态 31P NMR

• 批准号: 7012031
• 负责人: Mary E Hatcher-Skeers
• 金额: $ 17.02万
• 依托单位: CLAREMONT MC KENNA COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012031
• 关键词: DNA; binding sites; epigenetics; gene induction /repression; human; methylation; neoplasm /cancer; nucleic acid sequence; role; spine

DESCRIPTION (provided by applicant): Epigenetics is the inheritance of information based on gene expression. It determines when and where genetic information will be used. The most common human epigenetic modification is the methylation of cytosines in CpG steps of DNA sequences (Jones and Baylin 2002). The methylation state of these steps is known to play a crucial role in imprinting and gene silencing. This is tremendously important as methylation induced gene silencing is considered a precursor in many human cancers (Jones and Laird 1999). While much work has been done to understand the role of CpG methylation in tumorgenesis, the structural and dynamical effects of methylation are not well understood. Our research focuses on how proteins recognize specific DNA sequences and the effects of cytosine methylation on this recognition. In particular, we study the role of DNA dynamics in this recognition process. Our previous solid-state deuterium NMR studies of methylated and unmethylated DNA binding sites have shown that cytosine methylation quenches DNA backbone dynamics (Geahigan, Meints et al. 2000; Meints and Drobny 2001). However, these experiments required extensive sample preparation and allowed the study of only one nucleotide step at a time. We propose to use phosphorus NMR and complete lineshape analysis to investigate the backbone dynamics of each step in methylated and unmethylated DNA samples. This will allow us to rapidly probe a myriad of DNA binding sites for dynamical effects of cytosine methylation. The ability to quickly study a large number of binding sites will uncover any trends in methylation effects, which we hope can then be used to design tools for early detection of cancers. Oligonucleotide Sequences for 31P Dynamic NMR Study A. The EcoRI binding site, d(CGCGAATTCGCG)2. B. The CRE containing sites, d(ATGACGTCAT)2 and d(GAAAACGTTTTC)2. C. The cMyc binding site, d(ACCACGTGGT)2.

描述（由申请人提供）：表观遗传学是基于基因表达的信息遗传。它决定何时何地使用遗传信息。最常见的人类表观遗传修饰是 DNA 序列 CpG 步骤中胞嘧啶的甲基化（Jones 和 Baylin 2002）。已知这些步骤的甲基化状态在印记和基因沉默中起着至关重要的作用。这非常重要，因为甲基化诱导的基因沉默被认为是许多人类癌症的前兆（Jones 和 Laird 1999）。虽然已经做了很多工作来了解 CpG 甲基化在肿瘤发生中的作用，但甲基化的结构和动力学效应尚不清楚。 我们的研究重点是蛋白质如何识别特定的 DNA 序列以及胞嘧啶甲基化对这种识别的影响。我们特别研究 DNA 动力学在这一识别过程中的作用。我们之前对甲基化和非甲基化 DNA 结合位点的固态氘核磁共振研究表明，胞嘧啶甲基化会淬灭 DNA 主链动力学（Geahigan、Meints 等人，2000 年；Meints 和 Drobny，2001 年）。然而，这些实验需要大量的样品制备，并且一次只能研究一个核苷酸步骤。我们建议使用磷 NMR 和完整的线形分析来研究甲基化和非甲基化 DNA 样品中每个步骤的主链动力学。这将使我们能够快速探测大量 DNA 结合位点，以了解胞嘧啶甲基化的动态效应。快速研究大量结合位点的能力将揭示甲基化效应的任何趋势，我们希望随后可以将其用于设计癌症早期检测的工具。 用于 31P 动态 NMR 研究的寡核苷酸序列 A. EcoRI 结合位点，d(CGCGAATTCGCG)2。 B. 含有位点 d(ATGACGTCAT)2 和 d(GAAAACGTTTTC)2 的 CRE。 C. cMyc 结合位点，d(ACCACGTGGT)2。