Mechanistic investigation of protein translocation on DNA

化学系的生命过程化学项目正在支持加州大学圣巴巴拉分校的诺伯特·赖克教授研究细胞蛋白质如何在DNA上定位特定位置。众所周知，DNA存储了在细胞内以其他生物分子转录和翻译的遗传信息，这突显了每个细胞的生命。DNA信息的“阅读和解释”需要与细胞中的其他分子，特别是蛋白质之间的相互作用，并受其影响。通过DNA上特定位置的蛋白质进行搜索是具有挑战性的，因为DNA看起来非常一致。这项提议的重点是蛋白质如何识别DNA上的特定结合位置，然后它们如何在它们所结合的DNA上转移。这项工作将对科学家确定存储在DNA中的信息如何得到有效解释的能力产生更广泛的影响，这是所有细胞共同的过程。它正在对下一代科学家的教育产生进一步的影响，无论是本科生还是研究生，他们都有能力认识和解决与人类健康有关的科学问题。此外，赖克教授和他的学生积极开展活动，向广大受众解释科学是如何运作的，以及科学给社会带来了什么好处。作用于DNA的蛋白质负责DNA的绝大多数功能。这项建议的首要目标是提供对其中一种蛋白质--细菌DNA腺嘌呤甲基转移酶(DAM，修饰5‘-GATC-3’)的更深层次的了解。DAM和相关酶执行多个循环催化(过程催化)的能力对它们的生物学作用至关重要。然而，目前的过程催化模型在很大程度上是基于面临完全不同生物挑战的酶，没有考虑到这些酶的特征。例如，DNA甲基转移酶有效地修饰具有高度可变的位点间距离的多个识别位点，并且这种过程受到相互作用的蛋白质和位于靶点两侧的DNA序列的调节。本研究的目的是探讨DAM的过程催化作用的机制、调节及其在体内的重要性。将采取几种策略来解决广泛相关和悬而未决的问题，如Dam和其他蛋白质如何在超出常规模型预测的位置间距离与DNA底物进行连续作用；一些蛋白质，如Dam如何依靠片段内转移有效地移动长距离；以及单体Dam在不离开DNA的情况下修饰DNA链的机制是什么。

The Chemistry of Life Processes Program in the Division of Chemistry is supporting Professor Norbert Reich of University of California Santa Barbara to investigate how cellular proteins locate specific positions on DNA. It is well known that DNA stores genetic information that is transcribed and translated within cells in other biological molecules and that underscores the life of every cell. The "reading and interpretation" of the DNA information require and are modulated by interactions with other molecules in the cell, particularly proteins. The search by proteins of specific locations on DNA is challenging because DNA appears very uniform. This proposal focuses on how the proteins identify specific locations on DNA to bind to and then how they translocate on the DNA to which they are bonded. This work will have a broader impact on scientists' ability to determine how the information stored in DNA is productively interpreted, which is a process common to all cells. It is having further impact on the education of the next generation of scientists, both undergraduate and graduate students, capable of recognizing and addressing scientific issues relevant for human health. Furthermore, Professor Reich and his students actively engage in activities aimed at explaining to broad audiences how science works and what benefits science brings to society.Proteins that act on DNA are responsible for the vast majority of the functions of DNA. The overarching goal of this proposal is to provide a deeper understanding of one such protein, the bacterial DNA adenine methyltransferase (Dam, modifies 5'-GATC-3'). The ability of Dam and related enzymes to carry out multiple cycles of catalysis (processive catalysis) is critical to their biological roles. Yet, current models of processive catalysis are based largely on enzymes faced with entirely different biological challenges and do not account for the characteristics of these enzymes. For example, DNA methyltransferases efficiently modify multiple recognition sites with highly variable intersite distances, and this processivity is modulated by interacting proteins and DNA sequences that flank the target sites. The aims of the research are to investigate the mechanism, regulation, and in vivo importance of processive catalysis by Dam. Several strategies will be pursued to address broadly relevant and unanswered questions of how Dam and other proteins act processively with DNA substrates over intersite distances beyond the ones predicted by conventional models, how some proteins, such as Dam, rely on intra-segment transfer to efficiently move large distances, and what is the mechanism whereby the monomeric Dam modifies both strands of DNA without leaving the DNA.