Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function

脊椎动物核酸内切酶 G 对 5-羟甲基胞嘧啶识别和功能的结构适应

• 批准号: 2124202
• 负责人: Pui Ho
• 金额: $ 70.96万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2124202&HistoricalAwards=false
• 关键词: Structural; adaptation; vertebrate; endonuclease; hydroxymethylcytosine

The information encoded in DNA bases and various chemical modifications of the bases define different types of living organisms. The primary goal of this project is to determine how a DNA modification specific to vertebrate animals is used by a protein also found specifically in vertebrates, including humans, to help repair damaged DNA. The knowledge gained from this study will improve understanding of how seemingly small changes in the amino acid sequence of a protein through evolution can affect its structure and result in dramatic differences in how vertebrate versus invertebrate animals read and utilize modifications in their DNAs. In addition, this project will bring together biochemistry and art faculty to develop a new course on “Visual Communication in Science.” This course will engage science and art students in a collaborative environment to learn how to present scientific concepts and data in ways that are visually impactful and scientifically accurate and informative for a variety of audiences.5-Hydroxymethylcytosine (5hmC) is an epigenetic modification that serves multiple functions in vertebrates but has no known function in invertebrates. The project aims to determine how mouse endonuclease G (mEndoG) cleaves DNA at 5hmC sites in the context of four-stranded Holliday junctions to promote site-specific recombination. The roles of specific amino acids will be studied by swapping sequences between mEndoG and a worm homolog that does not cleave 5hmC-modified DNA. The resulting enzymes will be structurally characterized by X-ray crystallography and their functions examined by DNA binding and cleavage assays to test whether the sequence swaps result in corresponding changes in structures and functions between the vertebrate and invertebrate homologs. Finally, junction recognition by mEndoG will be studied to determine whether this enzyme is a 5hmC-specific junction resolvase or whether junctions mimic another substrate, such as the cross-over structures of DNA entering and exiting a nucleosome particle in chromatin fibers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

DNA碱基中编码的信息和碱基的各种化学修饰定义了不同类型的生物。该项目的主要目标是确定脊椎动物特有的DNA修饰如何被包括人类在内的脊椎动物特有的蛋白质用来帮助修复受损的DNA。从这项研究中获得的知识将提高人们对蛋白质氨基酸序列在进化过程中看似微小的变化如何影响其结构的理解，并导致脊椎动物和无脊椎动物在阅读和利用DNA中的修饰方面的巨大差异。此外，该项目还将把生物化学和艺术系联合起来，开发一门新的“科学中的视觉交流”课程。本课程将让理科和艺术专业的学生在协作的环境中学习如何以视觉上有影响力的、科学上准确的和信息量丰富的方式向各种受众展示科学概念和数据。5-羟甲基胞嘧啶(5hmC)是一种表观遗传修饰，在脊椎动物中具有多种功能，但在无脊椎动物中没有已知的功能。该项目旨在确定小鼠核酸内切酶G(MEndoG)如何在四链Holliday连接的背景下切割5hmC处的DNA，以促进位点特异性重组。特定氨基酸的作用将通过在mEndoG和不切割5hmC修饰的DNA的蠕虫同源物之间交换序列来研究。所产生的酶将通过X射线结晶学进行结构表征，并通过DNA结合和切割分析来检测它们的功能，以测试序列交换是否导致脊椎动物和无脊椎动物同源物之间的结构和功能发生相应的变化。最后，将研究mEndoG的连接识别，以确定该酶是否是5hmC特定的连接解析酶，或者连接是否模仿另一种底物，例如DNA进入和退出染色质纤维中的核小体颗粒的交叉结构。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。