Topologically complex DNA substrates to aid structure determination of the human topoisomerase IIa - Project Summary/Abstract Type II topoisomerases (TOP2s) are essenƟal enzymes that maintain DNA topology using an ATP-dependent “strand- passage” mechanism that physically moves one DNA segment through a transient, enzyme-mediated double-stranded break in another. Under normal condiƟons, TOP2s perform strand passage to facilitate fundamental processes such as DNA replicaƟon and transcripƟon; however, if improperly regulated, TOP2s also have the potenƟal to generate permanent DNA breaks, a property that has been exploited by successful chemotherapeuƟc drugs that “poison” TOP2 to induce cytotoxic DNA damage in cancer cells. MulƟple research approaches have helped characterize how TOP2 poisons bind the enzyme and stabilize key reacƟon intermediates that accompany DNA break formaƟon. Despite these advances, structural studies to date have only examined drug-bound TOP2s in complex with single, short DNA duplexes, precluding an understanding of how therapeuƟc agents interfere with the strand passage reacƟon on physiologically relevant DNA substrates such as chromaƟn and supercoils. Moreover, only one poison – etoposide – has been studied structurally in a naƟve context; all other drugs were imaged with TOP2 aŌer soaking into crystals pre-formed with etoposide, raising concerns that the binding mechanisms observed for these agents are arƟfactual and might differ substanƟally from what has been reported thus far. The objecƟve of the present applicaƟon is to capture currently elusive structural features of the TOP2 strand passage reacƟon on more naƟve-like substrates in the presence of different families of clinically used anƟ-TOP2 chemotherapeuƟcs. Human topoisomerase IIα (TOP2A), which is used by cells to support chromosome segregaƟon and cell proliferaƟon, will be used as a representaƟve model system. Strategies outlined in Aim 1 will employ chromaƟnized DNA substrates to beƩer understand how a large, disordered C-terminal domain (CTD) present in TOP2A helps to recruit the enzyme to mitoƟc chromaƟn, as well as how TOP2A may engage with the DNA entering and exiƟng a nucleosome. Binding assays using truncated TOP2A mutants and CTD pepƟdes will idenƟfy elements that are responsible for TOP2A’s interacƟon with nucleosomes, which will be imaged by cryo-EM. Aim 2 will use supercoiled minicircle DNAs and clinically deployed anƟ- TOP2 drugs for Ɵme-resolved cryo-EM studies to capture TOP2A as it is undergoing strand passage. This approach will not only enable a detailed structural analysis of elusive strand-passage intermediates, but also help resolve how diverse classes of TOP2 chemotherapeuƟcs poison the strand passage reacƟon to form toxic, double-stranded DNA breaks. Together, the proposed aims have the potenƟal to offer new mechanisƟc insights into the TOP2 strand passage reacƟon that will not only advance our understanding of how TOP2s safeguard genomic stability but will also unveil new possibiliƟes for exploiƟng the reacƟon for therapeuƟc benefit.
