CAREER: Structure and Function in Restriction Endonucleases

职业：限制性内切酶的结构和功能

• 批准号: 9875917
• 负责人: Cynthia Dupureur
• 金额: $ 38.8万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2001-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9875917&HistoricalAwards=false
• 关键词: CAREER; Structure; Function; Restriction; Endonucleases

Dupureur98759171. TECHNICALRestriction enzymes are the simplest biological agents ofsite-specific nucleic acid chemistry and as such serve as molecularlaboratories for dissecting natural activities and engineering new ones.In spite of the importance of the metal ion dependence of restrictionenzyme mechanisms, it is unclear how many metal ions bind these enzymes. In order to propose relevant reaction mechanisms, detailed measurements of metal ion affinity, stoichiometry, and substrate coordination in restriction enzymes must first be developed and conducted in a direct and comprehensive fashion. As evidenced by the null activity of enzymes mutated distal to the active site, metal ion-mediated catalysis alsoappears to be tightly coupled to conformational changes, which prompted the hypothesis that these mutations compromise critical metal ion-substrate interactions in the active site. This study applies an unique combination of instrumental methods and international, industrial, and academic collaborations to the issues of metal ion binding and conformational behavior in restriction endonucleases. To better understand underlying themes in endonuclease function, two representative enzymes will be compared. The first specific aim of this study is to determine the affinities and binding stoichiometries for required divalent metal ions. These experiments are coupled to studies of the importance of metal ion-substrate interactions in the active site, using a combination of isothermal titration calorimetry (ITC), site-directed mutagenesis, phosphorothioate substitution, and paramagnetic resonance methods. The staggered-end cutter EcoRI endonuclease and the blunt-end cutter PvuII endonuclease serve as the representative restriction endonucleases. The second specific aim is to examine the extent and nature of metal ion and substrate-induced conformational changes in restriction enzymes. Isotopic labeling and multidimensional nuclear magnetic resonance (NMR) methods are applied to PvuII endonuclease, the smallest type II restriction enzyme yet characterized. Parallel to the execution of research plans, two advanced graduate courses will be developed. The first course introduces advanced spectroscopic methods to the study of protein structure and function and serves a growing and vital biophysical chemistry community on campus. Complementary to this is a course which develops the scientific writing skills among advanced biochemistry graduate students through a discussion of both the structure and language of proposals and involves the participation of both academic and industrial collaborators.2. Non-technical Restriction enzymes are proteins that recognize and cut specificsequences of DNA with the assistance of metal ions. These "molecularscissors" are powerful tools of biotechnology. There is considerableinterest in expanding the repertoire of these enzymes, that is, developing new enzymes with new specificities. The approach of this study is to understand how existing enzymes work. This study is divided into parts: First, to understand how the metals ion help the enzyme cut DNA, comparing the metal ion binding properties of two representative restriction enzymes, PvuII endonuclease and EcoRI endonuclease. Instrumental methods are applied to determine how many metal ions bind each enzyme, where they bind, and how the enzyme-bound metal ions interact with the DNA. This helps us understand what is required for DNA cutting. The question of how the conformation or shape of PvuII endonuclease changes when it interacts with metal ions and different DNA sequences is also studied. To accomplish this, nuclear magnetic resonance (NMR) spectroscopy, an instrumental method which is sensitive to slight changes in enzyme conformation is applied. Together, these two aims help us understand how DNA sequence recognition and metal ion-assisted DNA cleavage are related in these enzymes. Concurrent with and complementary to these research activities, two new elective graduate courses will be developed. The first is a survey of spectroscopic methods as applied to problems in protein structure-function and will involve faculty members with expertise in this area. The other elective is a course in the structure and language of research proposals and will include discussions led by study sectionmembers and scientists from industry.

Dupureur98759171。限制性内切酶是位点特异性核酸化学的最简单的生物制剂，作为解剖自然活动和设计新活动的分子实验室。尽管限制性酶机制中金属离子依赖性很重要，但目前尚不清楚有多少金属离子结合这些酶。为了提出相关的反应机制，必须首先开发并以直接和全面的方式进行限制性内切酶中金属离子亲和力、化学计量学和底物配位的详细测量。正如活性位点远端突变的酶的零活性所证明的那样，金属离子介导的催化也似乎与构象变化紧密耦合，这促使了这些突变损害活性位点中关键的金属离子-底物相互作用的假设。本研究将仪器方法和国际、工业和学术合作的独特结合应用于限制性内切酶的金属离子结合和构象行为问题。为了更好地理解内切酶功能的基本主题，将比较两种具有代表性的酶。本研究的第一个具体目的是确定所需二价金属离子的亲和力和结合化学计量学。这些实验与金属离子-底物相互作用在活性位点的重要性的研究相结合，使用等温滴定量热法（ITC）、位点定向诱变、硫代酸替代和顺磁共振方法的组合。交错端切刀EcoRI内切酶和钝端切刀PvuII内切酶是具有代表性的限制性内切酶。第二个具体目的是检查的程度和性质的金属离子和底物诱导构象变化的限制性内切酶。采用同位素标记和多维核磁共振（NMR）方法对PvuII内切酶进行了研究，该酶是目前已知的最小的II型限制性内切酶。在执行研究计划的同时，还将开设两门高级研究生课程。第一门课程介绍了先进的光谱方法来研究蛋白质的结构和功能，并为校园内不断发展和重要的生物物理化学社区服务。与此相辅相成的是一门课程，该课程通过讨论提案的结构和语言来培养高级生物化学研究生的科学写作技能，并涉及学术和工业合作者的参与。非技术限制性内切酶是在金属离子的帮助下识别和切割特定DNA序列的蛋白质。这些“分子剪刀”是生物技术的有力工具。有相当大的兴趣扩大这些酶的剧目，即开发新的酶与新的特异性。这项研究的方法是了解现有的酶是如何工作的。本研究分为以下几个部分：首先，了解金属离子如何帮助酶切割DNA，比较两种具有代表性的限制性内切酶PvuII内切酶和EcoRI内切酶的金属离子结合特性。仪器方法应用于确定有多少金属离子结合每种酶，它们结合在哪里，以及酶结合的金属离子如何与DNA相互作用。这有助于我们了解DNA切割需要什么。还研究了PvuII核酸内切酶与金属离子和不同DNA序列相互作用时的构象或形状变化。为了做到这一点，核磁共振（NMR）光谱，一种仪器方法，是敏感的酶构象的微小变化应用。总之，这两个目标帮助我们了解DNA序列识别和金属离子辅助DNA切割在这些酶中是如何相关的。与这些研究活动同时进行并互为补充的是，将开设两门新的研究生选修课程。第一个是光谱方法应用于蛋白质结构-功能问题的调查，并将涉及具有该领域专业知识的教师。另一门选修课是关于研究提案的结构和语言的课程，将包括由研究小组成员和工业界科学家领导的讨论。