Structure and Function of DNA topoisomerase IIB
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
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.
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.
