Indirect Readout and Specificity in DNA Binding Enzymes

描述(由申请人提供)：识别DNA，包括序列和结构特异性，对细胞的健康功能非常重要，从复制到转录，再到DNA修复。这一过程的失败可能会导致严重的疾病，包括癌症。序列和结构特异的DNA识别都有一个共同的机制，涉及利用DNA固有的构象能量学，这是由于不同序列之间的优先碱基堆积作用，或者是由于双链结构的化学和非化学修饰。这里描述的项目旨在研究DNA结合酶中序列特异性的分子机制以及这种特异性的松弛。在这类研究中使用酶比使用非酶DNA结合蛋白更有利，因为由于过渡态相对于基态络合物的结构更严格，热力学参数可以更准确地提取。这些研究没有关注蛋白质-DNA的直接相互作用，而是解决了微妙和更复杂的识别机制，包括利用DNA的序列依赖构象偏好和由于变构调节而在蛋白质内发生的构象变化。选定的II型限制性内切酶将用于研究，因为它们具有无与伦比的特异性、活性、多样性和实验实用性。在研究中将结合使用X射线结晶学的三维结构测定以及动力学和结合亲和力测量。

DESCRIPTION (provided by applicant): Recognition of DNA, both sequence and structure specific, is important in the healthy functioning of the cell, from replication, to transcription, to DNA repair. Failure of this process can lead to severe disease, including cancer. Both sequence and structure specific DNA recognition share a common mechanism involving the utilization of the intrinsic conformational energetics of DNA due, either to preferential base stacking interactions of different sequences, or to chemical and non-chemical modifications of the duplex structure. The project described herein is designed to investigate the molecular mechanisms of sequence specificity, and the relaxation of this specificity, in DNA binding enzymes. The use of enzymes in such studies is advantageous over the use of non-enzymatic DNA binding proteins, as thermodynamic parameters can be extracted more faithfully as a result of the greater structural stringency of the transition state relative to the ground state complex. Rather than focusing on direct protein-DNA interactions, the investigations address subtle and more complex mechanisms of recognition, including the utilization of sequence dependent conformational preferences of the DNA and conformational changes occurring within a protein as a result of allosteric modulation. Selected type II restriction endonucleases will be used in the study, due to their unparalleled specificity, activity, diversity, and experimental practicality. A combination of three dimensional structure determinations using x-ray crystallography, as well as kinetic and binding affinity measurements, will be used in the investigations.