Structure and Function of DNA topoisomerase IIB

DNA拓扑异构酶IIB的结构和功能

• 批准号: 2143143
• 金额: --
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2143143
• 关键词: Structure; Function; DNA; topoisomerase; IIB

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.

使用生化测定、生物物理测定和晶体学对 DNA 拓扑异构酶 II beta 进行生化和结构分析。该酶参与转录调节。 II 型 DNA 拓扑异构酶是必需的；它们通过以受控方式断裂和重新连接 DNA 主链来催化 DNA 中的拓扑重排。 II 型拓扑异构酶的毒物可以优先靶向原核和真核酶，使其成为人类有吸引力的候选药物和细菌抗感染药物。人类 TOP2B 已在转录起始中实现，两种亚型在增殖细胞中均高度表达。了解 TOP2B 的活性和调节对于进一步了解这两种亚型并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 个具有酶功能的耐药突变。除 H514Y 和 A596T 外，所有其他蛋白均已表达并进行生化表征（Leontiou 等人，2004、2006、2007 和 Gilroy 等人，2006）。这些突变大多数位于核心催化结构域内（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 的弛豫活性增加了 50%，分解活性降低了 50%。该区域显然对于催化至关重要，但泛素化在 TOP2 催化活性中的作用尚未完全了解。据报道，类似的翻译后修饰 SUMO 化可以改变 TOP2 的活性，并且 SUMO 修饰位点存在于 TOP2A 核心中直接参与结合 G 段的残基上（Wendorff 等人，2012）。在本次奖学金中，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 Caroline Austin 小组拥有一个成熟的酵母表达系统，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在细菌中轻松表达，并且该构建体已被证明适用于 X 射线晶体学（Wu 等人，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。 Tim Blower 之前曾研究过拓扑异构酶，包括 TOP2B，他将在达勒姆对学生进行 X 射线晶体学培训。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。