Topoisomerase II beta in the cellular response to DNA damage

我们的DNA有大约30亿个碱基对，编码了细胞在其生命周期内的所有结构和功能的蓝图。保持DNA的完整性和稳定性对细胞和整个生物体的生存至关重要。随着每一次细胞分裂，这种DNA必须被忠实地复制并平均分配到子细胞中。它还必须准备好对任何DNA破坏事件做出反应。如果做不到这一点，可能会导致DNA突变的积累，细胞衰老或细胞死亡。令人惊讶的是，活着的有机体中的每个细胞每天都要忍受数以万计的DNA侮辱。它们自发地产生于内源(来自体内)和外源(来自体外)。矛盾的是，氧虽然对生命是绝对必要的，但作为细胞新陈代谢的副产品，它会转化为活性氧物种；这些会以多种方式改变DNA，有可能导致突变或DNA片段断裂。在地壳中发现的自然辐射，以及来自太空、航空旅行和医疗X光的暴露，都可能导致我们的DNA断裂。值得庆幸的是，已知有200多个哺乳动物基因参与了DNA损伤的检测和修复。也许矛盾的是，其他对细胞功能至关重要的蛋白质可以对DNA损伤做出反应，并被困在DNA上。这是一种潜在的危险，因为这些蛋白质可能会增加损害负担，或者减缓受损部位的修复。这些蛋白质行为的特征是一个需要深入研究的领域。这一应用通过研究拓扑异构酶II(TOP2)的这种行为来为该领域做出贡献。*拓扑异构酶是一个进化保守的酶家族，参与解开和解开DNA，以允许其复制(复制)和分离(分离)到子细胞中。它们存在于所有生物体中，包括细菌、病毒、植物和动物。虽然拓扑异构酶(包括TOP2形式)已经被广泛研究，但来自我的实验室的新数据表明，这种蛋白质在对辐射造成的损害的反应中扮演着尚未确定的角色。在我们的实验室，我们已经开发了实验工具，使我们能够确定TOP2在细胞对辐射的反应中所扮演的角色。利用这些独特的试剂，再加上通过国际合作获得的额外资源，我们将专门调查TOP2是如何被困在DNA上的。利用TOP2的工程突变形式，我们将阐明酶活性的要求并研究其运动的动力学。*破译我们的细胞如何对DNA损伤做出反应，以及识别和表征对这种损伤做出反应的新蛋白质，对于了解广泛生物中的DNA修复以及识别有助于保持DNA长期稳定性的试剂的开发目标具有重要意义。

Our DNA, with ~3 billion base pairs, encodes the blueprint' for all the structures and functions of a cell over its lifespan. Maintaining the integrity and stability of this DNA is of paramount importance to survival of the cell and the entire organism. With each cell division, this DNA must be faithfully copied and equally distributed into daughter cells. It must also be prepared to react to any DNA damaging events. Failure to do so can lead to the accumulation of mutations in our DNA, cell aging or cell death. Astoundingly, each cell in a living organism endures tens of thousands of DNA insults every day. These arise spontaneously from both endogenous (from within the body) and exogenous (from outside the body) sources. Paradoxically, oxygen, although absolutely essential for life, is converted to reactive oxygen species as by-products of cell metabolism; these can alter DNA in numerous ways, potential leading to mutations or broken DNA fragments. Naturally occurring radiation found in the earth's crust and exposure from space, airline travel and medical x-rays can lead to breaks in our DNA. Thankfully, over 200 mammalian genes are known to be involved in the detection and repair of DNA damage. Perhaps paradoxically, other proteins critical for cell function can react to DNA damage and become trapped on the DNA. This is a potential hazard, as these proteins could add to the damage burden, or slow the repair of damage sites. Characterization of these protein behaviours is an area of intense investigation. This application contributes to the field by investigating this behaviour for one enzyme, topoisomerase II (TOP2).******The topoisomerases are an evolutionarily conserved family of enzymes that are involved in untangling and unwinding DNA to allow for its replication (copying) and segregation (separation) into daughter cells. They are found in all organisms, including bacteria, viruses, plants and animals. Although topoisomerases (including the TOP2 form) have been extensively studied, emerging data from my laboratory points to an as yet uncharacterized role for this protein in the response to damage caused by radiation.******In our laboratory, we have developed experimental tools that position us to determine the role TOP2 plays in the cell's response to radiation. Using these unique reagents, together with additional resources obtained through international collaborations, we will specifically investigate how TOP2 becomes trapped on DNA. Using engineered mutant forms of TOP2, we will clarify the requirement for enzyme activity and study the kinetics of its movement.******Deciphering how our cells respond to the DNA damage, and identification and characterization of new proteins that respond to this damage, is of importance in understanding DNA repair in a wide range of organisms and for identifying targets for the development of agents that can help preserve the long-term stability of our DNA.