Structure and Function of DNA topoisomerase IIB

DNA拓扑异构酶II β的生化和结构分析，使用生化分析，生物物理分析和晶体学。这种酶参与转录调节。II型DNA拓扑异构酶是必不可少的；它们通过以一种受控的方式破坏和重新连接DNA主干来催化DNA的拓扑重排。II型拓扑异构酶的毒素可以优先靶向原核和真核酶，使其成为人类和细菌抗感染的有吸引力的候选药物。人类TOP2B已在转录起始中实现，这两种亚型在增殖细胞中都高度表达。了解TOP2B的活性和调控对于进一步了解这两种亚型和产生更有针对性的治疗方法至关重要。在之前的酵母筛选中，我们在TOP2B中选择了9个酶功能耐药突变。除了H514Y和A596T外，所有的基因都已表达并进行了生化鉴定（Leontiou等人，2004,2006,2007和Gilroy等人，2006）。这些突变大部分位于核心催化结构域（残基400-1200），G550、H514和A596紧邻经常泛素化的赖氨酸（GGbase https://ggbase.hms.harvard.edu/）。TOP2B G550R的弛豫活性提高50%，十癸烯化活性降低50%。该区域显然对催化至关重要，但泛素化在TOP2催化活性中的作用尚未完全了解。有报道称，类似的翻译后修饰SUMO修饰可以改变TOP2的活性，并且SUMO修饰位点存在于TOP2A核心直接参与结合g段的残基上（Wendorff et al, 2012）。在本研究中，我们计划研究H514Y和A596T对TOP2B生化功能的影响，并将这些突变蛋白与泛素化靶位点K515、K551和K595的突变进行比较。纽卡斯尔的卡洛琳·奥斯汀的研究小组已经建立了一个酵母表达系统，可以表达足够数量的TOP2B蛋白，用于生化和结构研究。TOP2B的催化核心结构域可以很容易地在细菌中表达，这种结构已经被证明适用于x射线晶体学（Wu et al, 2011）。体外泛素化系统将用于修饰核心蛋白，以确定泛素化对蛋白质功能和结构的影响。蒂姆·布洛尔（Tim Blower）之前曾研究过拓扑异构酶，包括TOP2B，他将在达勒姆大学（Durham）培训这名学生x射线晶体学。纽卡斯尔的Bert Van Den Berg带来了翻译后修饰后蛋白质结构变化的专业知识。

Biochemical and structural analysis of DNA topoisomerase II beta, using biochemical assays, biophysical assays and crystallography. This enzyme is involved in transcriptional regulation. Type II DNA topoisomerases are essential; they catalyse topological rearrangements in DNA by breaking and re-joining the DNA backbone in a controlled manner. Poisons of type II topoisomerases can target prokaryotic and eukaryotic enzymes preferentially, making them attractive drug candidates in humans and anti-infectives in bacteria. Human TOP2B has been implemented in transcriptional initiation, both isoforms are highly expressed in proliferating cells. Understanding the activity and regulation of TOP2B is vital for understanding the two isoforms further and generating more targeted therapeutics. In a previous yeast screen, we selected nine enzymatically functional drug-resistant mutations in TOP2B. All but H514Y and A596T have been expressed and biochemically characterised (Leontiou et al, 2004, 2006, 2007 and Gilroy et al, 2006). The majority of these mutations are located within the core catalytic domain (residues 400-1200) and G550, H514 and A596 are immediately adjacent to lysines that are frequently ubiquitinated (GGbase https://ggbase.hms.harvard.edu/). The relaxation activity of TOP2B G550R is increased by 50% and decatenation activity is reduced by 50%. This region is clearly vital for catalysis and yet the role of ubiquitination in the catalytic activity of TOP2 is not fully understood. A similar post-translational modification, SUMOylation, has been reported to alter the activity of TOP2, and a SUMO modification site is present in the core of TOP2A on a residue directly involved in binding the G-segment (Wendorff et al, 2012). In this studentship we plan to investigate the effect of H514Y and A596T on the biochemical functions of TOP2B, and to compare these mutated proteins with mutations at the ubiquitination target sites K515, K551 and K595. Caroline Austin's group in Newcastle have an established yeast expression system that can express TOP2B proteins in quantities sufficient for biochemical and structural studies. The catalytic core domain of TOP2B can be expressed readily in bacteria and this construct has already proven suitable for X-ray crystallography (Wu et al, 2011). An in vitro ubiquitination system will be used to modify the core proteins to determine the effect of ubiquitination on both the function and structure of the protein. Tim Blower has worked with topoisomerases previously, including TOP2B, and he will train the student in X-ray crystallography in Durham. Bert Van Den Berg in Newcastle brings expertise on structural changes in proteins following post translational modifications.