Upcoming Genetics & Molecular Biology Cells, Biochemistry & Physiology

Reflections on DNA - how do topoisomerases distinguish between left-handed and right-handed superhelices?

Funder BBSRC (part of UKRI)
Recipient organisation University of Glasgow
Funding Not disclosed
Period Oct 2028 — Oct 2028

In plain English

AI plain-English summary

Every time a cell copies its DNA, the long double helix gets twisted into knots and tangles that must be undone by enzymes called topoisomerases. These enzymes make controlled breaks in DNA, pass another strand through the gap, and reseal it—a process that is essential for life and also a target for antibiotics and cancer drugs. But topoisomerases face a subtle challenge: DNA superhelices can coil in either a left-handed or right-handed direction, and the enzymes must tell them apart to work correctly. No one knows exactly how they do this. This project aims to build artificial DNA catenanes—interlocked rings like molecular chain links—with precisely defined left- or right-handed twists. Using enzymes called serine integrases, the researchers will create these chiral substrates in high yield, then hand them to different type II topoisomerases to see how each enzyme responds. They will also capture snapshots of the enzymes in action using cryo-electron microscopy. This is fundamental science. Understanding how topoisomerases distinguish chirality could eventually guide the design of more selective drugs that block these enzymes in bacteria or cancer cells without harming healthy human DNA.

View original technical description
DNA is a long double-helical molecule that becomes knotted, tangled and over- or under-twisted during transcription, replication, recombination and other cellular processes. All DNA-based lifeforms require topoisomerases, enzymes that unknot, untangle and relax torsional stress in the DNA. Type II topoisomerases make transient double-strand breaks through which another DNA segment is passed to allow unlinking and unknotting of DNA, as well as to relax over- and under-winding. Topoisomerases are important drug targets for anti-bacterial and anti-cancer agents. The overarching aims of this project are to generate catenated substrates with defined structure and chirality and to use these to study the mechanism of different type II topoisomerases. The specific aims are: 1) To use large serine integrases to make both left-handed and right-handed catenanes. 2) With our industrial collaborator, to develop techniques using large serine recombinases and Xer site-specific recombination to generate and purify novel chiral substrates for topoisomerases at high yield. 3) To use these substrates to study the mechanism by which different type II topoisomerases recognize the chirality of DNA knots and catenanes and discriminate between knotting/catenation and supercoiling. 4) To use defined catenated or knotted DNA substrates to capture the structures of type II topoisomerases in action using cryp electron microscopy.

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