Indirect Readout and Specificity in DNA Binding Enzymes

描述（由申请人提供）：DNA识别，包括序列和结构特异性，在细胞的健康功能中很重要，从复制，转录到DNA修复。这一过程的失败可能导致严重的疾病，包括癌症。序列特异性和结构特异性DNA识别都有一个共同的机制，包括利用DNA固有的构象能量，这要么是由于不同序列的优先碱基堆叠相互作用，要么是由于双工结构的化学和非化学修饰。本文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。在此类研究中使用酶比使用非酶DNA结合蛋白更有利，因为相对于基态复合物，过渡态的结构更严格，因此可以更忠实地提取热力学参数。而不是专注于直接的蛋白质-DNA相互作用，研究解决了微妙和更复杂的识别机制，包括利用序列依赖的DNA构象偏好和构象变化发生在蛋白质内作为变构调节的结果。所选的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。使用x射线晶体学的三维结构测定，以及动力学和结合亲和力测量，将用于研究。

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.