Selectivity determinants in DNA restriction and modification enzymes

DNA 限制酶和修饰酶的选择性决定因素

基本信息

批准号：
7325813
负责人：
JOHN J. PERONA
金额：
$ 27.71万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-03-01 至 2009-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7325813
关键词：
Active Sites Address Adenine Affect Amino Acids Area Base Pairing Binding Biological Assay Chemicals Chemistry Cleaved cell Closure Color Complement Complex Coupled DNA DNA Binding DNA Modification Methylases DNA Modification Process DNA Repair Enzymes DNA Restriction Enzymes DNA Restriction-Modification Enzymes DNA Sequence DNA Sequence Rearrangement DNA modification methylase EcoRI Data Decompression Sickness Deoxyribonuclease I Dependency Electrostatics Engineering Enzymes Equilibrium Exhibits Family Fluorescence Fluorescence Resonance Energy Transfer Fostering Generations Genetic Engineering Goals Ions Kinetics Measurement Mediating Metals Methionine Methods Methyltransferase Modification Molecular Mutation Nucleic Acids Object Attachment Pathway interactions Play Positioning Attribute Process Proteins RNA Rate Reaction Roentgen Rays Role Site Site-Specific DNA Methyltransferase (Cytosine-Specific)Specificity Structure System Techniques Testing Topoisomerase Type II site-specific deoxyribonuclease Variant X-Ray Crystallography analog base biological research cofactor conformational conversion endodeoxyribonuclease EcoRV endonuclease enzyme model human disease inorganic phosphate intercalation interest methylphosphonate mutant prototype recombinase research study restriction enzyme stopped-flow fluorescence therapy development three dimensional structure tool

项目摘要

The detailed structural and energetic origins of sequence-specificity in the recognition and modification of DNA will be investigated using prokaryotic restriction endonucleases and modification methylases as experimental systems. First, the specificity-determining induced fit conformational change in the best-studied EcoRV endonuclease will be examined using stopped-flow fluorescence techniques, chemical quench-flow kinetics, and Xray crystallography. These approaches will be applied to the study of noncognate complexes and to enzyme variants, to test current hypotheses regarding the origins of sequence selectivity. The mechanism of DNA bending by this enzyme will also be tested by modifications to both enzyme and DNA, coupled to functional and structural analyses. Next, the diversity of metal-dependent cleavage chemistries in three other structurally well-characterized type II restriction endonucleases will be studied by enzymological techniques. {{Finally, we shall extend our inquiry into the induced-fit origins of sequence selectivity by exploring how the "base-flipping" structural transitions in both adenine and cytosine DNA methylases play crucial roles in mediating the approach to the transition state in cognate and noncognate complexes.}} Elucidation of how DNA sequence recognition is manifested through induced-fit conformational rearrangements in the restriction endonuclease and methylase families will be relevant to understanding this phenomenon in a variety of nucleic-acid modifying enzymes including recombinases, topoisomerases, and enzymes of DNA repair and RNA modification pathways. Further, a clearer appreciation of the detailed structural and energetic basis for specificity and rate enhancement forms an essential basis for the rational engineering of enzymes with new specificities. Since restriction endonucleases and DNA methylases are an essential part of the recombinant DNA technology that underlies all areas of biological research, this is a prospect of substantial importance to the development of therapies for many human diseases.

在识别和修改中序列特异性的详细结构和能量起源 DNA将使用原核限制性核酸内切酶和修饰甲基酶进行研究实验系统。首先，确定的特异性诱导的拟合构象变化是最好的研究 ECORV核酸内切酶将使用停止流量荧光技术，化学淬灭流。动力学和X射线晶体学。这些方法将应用于非共同复合物的研究以及酶变体，以测试有关序列选择性起源的当前假设。这该酶的DNA弯曲机制也将通过对酶和DNA的修饰进行测试，耦合到功能和结构分析。接下来，在金属依赖性乳沟化学的多样性将通过酶学研究其他三个结构上良好的II型限制性核酸内切酶技术。 {{最后，我们将调查扩展到序列选择性的诱导拟合起源探索腺嘌呤和胞嘧啶DNA甲基酶的“碱基化”结构过渡如何在介导对同源和非共同复合物中过渡状态的方法中的关键作用。}}} 通过诱导拟合构象表现出DNA序列识别方式的阐明限制性核酸内切酶和甲基酶家族的重排将与理解这一点有关各种核酸修饰酶的现象，包括重物组织酶，拓扑异构酶和 DNA修复和RNA修饰途径的酶。此外，对详细信息更清楚地欣赏特异性和增强率的结构和能量基础是理性的基础具有新特异性的酶的工程。由于限制性核酸内切酶和DNA甲基酶是一种重组DNA技术是生物学研究所有领域的基础，这是一个对于许多人类疾病的疗法发展具有重要意义的前景。