Tn7 Transpostion into DNA Replication/Repair Complexes

描述(由申请人提供)：该实验室的长期目标是了解生物体如何在分子水平上维护他们的基因组信息。我们正在研究一种在称为转座的过程中改变基因组的方法。转座子是一种可移动的DNA元件，可以在细胞内移动，几乎在所有有机体中都有，在人类基因组中也很丰富。为了在分子水平上更好地理解转座，我们使用了细菌转座子Tn7。Tn7转座特别令人感兴趣，因为它还允许我们了解DNA复制和修复，因为Tn7识别这些过程中涉及的复合体作为插入目标。这项工作与人类健康有关，因为它将使人们更好地了解在所有活着的有机体中发现的细胞DNA修复功能，这些功能对预防癌症非常重要。Tn7能够识别与多种形式的DNA代谢相关的结构或复合体，使用TnsE编码的蛋白质。TnsE介导的转座优先发生在能够在细胞之间移动的质粒中，称为结合质粒。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复制终止的转座。我们将确定TnsE是否直接与β-钳制加工性因子相互作用来识别滞后链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。转座途径将在重组的体外反应中建立，以在分子水平上了解靶点选择的机制。与公共卫生相关：我们将使用一个高度易驯化的细菌系统，以更好地了解DNA复制和修复的功能。这将提供非常必要的洞察，了解人类中非常相似的系统，当这些系统不起作用时，可能会导致癌症。这将服务于公众健康，因为对DNA修复和复制的更好理解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.