Tn7 Transpostion into DNA Replication/Repair Complexes

描述（由申请人提供）：实验室的长期目标是了解生物体如何在分子水平上维持其基因组信息。我们正在研究一种可以改变基因组的方法，这个过程被称为转位。转座子是一种可移动的DNA元件，可以在细胞内移动，几乎存在于所有生物体中，并且在人类基因组中含量丰富。为了更好地理解分子水平上的转座子，我们正在使用细菌转座子Tn7。Tn7转位是特别有趣的，因为它也允许我们理解DNA复制和修复，因为Tn7识别参与这些过程的复合物作为插入目标。这项工作与人类健康有关，因为它将使我们更好地了解在所有生物体中发现的细胞DNA修复功能，这些功能对预防癌症很重要。Tn7能够使用Tn7编码的蛋白TnsE识别与多种形式的DNA代谢相关的结构或复合体。tnse介导的转座优先发生在能够在细胞之间移动的质粒中，称为共轭质粒。tnse介导的途径也识别与DNA双链断裂修复相关的结构，并允许Tn7优先指导DNA复制终止的转位。我们将确定TnsE是否直接与β钳加工因子相互作用以识别滞后链DNA复制。我们将确定TnsE蛋白如何与靶DNA和核心机制相互作用，以招募转座装置的其余部分。在体外反应重构中建立转位途径，在分子水平上了解靶位选择的机制。与公共卫生相关：我们将使用高度易处理的细菌系统来更好地了解DNA复制和修复功能。这将为人类非常相似的系统提供急需的见解，当这些系统不起作用时可能导致癌症。这将有利于公众健康，因为对DNA修复和复制的更好理解将有助于深入了解癌症的起源。

DESCRIPTION (provided by applicant): The long-term goal of the lab is to understand how organisms maintain their genomic information at the molecular level. We are investigating one of the ways genomes can be altered in a process called transposition. Transposons are mobile DNA elements that can move within a cell which are found in nearly every organism and are abundant in the human genome. To better understand transposition at the molecular level we are using the bacterial transposon Tn7. Tn7 transposition is of particular interest in that it also allows us to understand DNA replication and repair because Tn7 recognizes complexes involved in these processes as insertion targets. This work is related to human health because it will lead to a better understanding of cellular DNA repair functions found in all living organisms that are important in preventing cancer. Tn7 is able to recognize a structure or complex associated with multiple forms of DNA metabolism using the Tn7-encoded protein TnsE. TnsE-mediated transposition preferentially occurs into plasmids capable of moving between cells called conjugal plasmids. The TnsE-mediated pathway also recognizes structures associated with the repair of DNA double-strand breaks and allows Tn7 to preferentially direct transposition where DNA replication terminates. We will determine if TnsE interacts directly with the beta-clamp processivity factor to recognize lagging- strand DNA replication. We will determine how the TnsE protein interacts with a target DNA and core machinery to recruit the rest of the transposition apparatus. The transposition pathway will be established in a reconstituted in vitro reaction to understand the mechanism of target site selection at the molecular level. Relevance to Public Health: We will use a highly tractable bacterial system to get a better understanding of how DNA replication and repair function. This will provide much needed insight into very similar systems in humans which when non-functional can lead to cancer. Public health will be served because a better understanding of DNA repair and replication provides insight into the genesis of cancer.