DNA

U of T researchers discover intricate process of DNA repair in genome stability

Christopher.Sorensen — Thu, 06 Feb 2020 16:39:42 +0000

U of T researchers discover intricate process of DNA repair in genome stability

Christopher.Sorensen Thu, 02/06/2020 - 11:39

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Roxanne Oshidari and Karim Mekhail, both in the Faculty of Medicine, worked closely with colleagues in the Faculty of Applied Science & Engineering to understand aspects of the DNA repair process (photo courtesy of Faculty of Medicine)

Jim Oldfield

Topic

Breaking Research

DNA

Faculty of Applied Science & Engineering

Faculty of Medicine

Graduate Students

Laboratory Medicine and Pathobiology

Research & Innovation

An elaborate system of filaments, liquid droplet dynamics and protein connectors enables the repair of some damaged DNA in the nuclei of cells, researchers at the University of Toronto have found.

The findings further challenge the belief that broken DNA floats aimlessly – and highlight the value of cross-disciplinary research in biology and physics.

DNA repair helps ensure genome stability, which in turn allows cells to function and promotes health in all organisms. Double-strand DNA breaks are especially toxic to cells, and researchers had assumed for decades that these breaks floated inside cell nuclei without direction, until they trigger other cellular changes or happen on a fixer mechanism.

That thinking began to change in 2015, when Karim Mekhail, an associate professor of laboratory medicine and pathobiology in U of T’s Faculty of Medicine, and his lab showed that damaged DNA can be intentionally transported by motor protein “ambulances” to DNA “hospitals,” areas enriched with certain repair factors in the nuclei. The researchers later worked with U of T aerospace engineers to show that after a single double-strand break, DNA travels for repair via long “autobahns” of thread-like microtubules, which are also moving.

In the current study, Mekhail and lead author Roxanne Oshidari, a PhD candidate, looked at yeast cells with many DNA double-strand breaks and showed that co-ordination between shorter types of microtubule filaments and liquid-like droplets composed of DNA repair proteins enables the creation and function of a DNA repair centre.

“The liquid droplets work with intranuclear microtubules to promote the clustering of damaged DNA sites,” says Mekhail. “Repair proteins at these different sites assemble in droplets that fuse into a larger repair-centre droplet through the action of the shorter nuclear microtubules.”

This larger oil-like droplet then behaves like a spider, shooting out a web of star-shaped filaments that tether to the longer “autobahns” along which damaged DNA can be transported to the DNA hospitals, Mekhail says.

The findings were recently published in the journal Nature Communications.

Mekhail turned to Nasser Ashgriz, a professor in U of T’s department of mechanical and industrial engineering in the Faculty of Applied Science & Engineering, to measure and understand the role of droplets in the repair process. “You couldn’t ask for better expertise in fluid dynamics, and he was just across the road,” Mekhail says of Ashgriz, who runs U of T’s multi-phase flow and spray systems lab.

Mekhail brought a video of the droplets to Ashgriz, who projected it on a large screen in his office and confirmed that fluid dynamics appeared to be at play. But communication across the biology-physics divide was challenging. “Understanding what they do was very difficult in the beginning because our terminologies are totally different,” says Ashgriz.

When he and Mekhail used plain language to describe how the droplets behaved, however, things started to make sense. “We focused on the physical aspects of the droplets,” Ashgriz says. “The physics that cause their motion and dynamics became our common language.”

After months of discussions and experiments, computer simulations repeatedly predicted that the shorter filaments would move like pistons, lowering pressure in the nucleoplasm and creating a suction effect that leads to the fusion of droplets. Mekhail and his team confirmed that finding in their lab.

“Often when we dive deep in the specifics of a field, we get separated from one another,” Ashgriz says. “Bringing together people with different views can really improve understanding, and this work was a good example – with credit to Karim for his vision and initiative.”

Mekhail and his team also uncovered further important properties of the repair droplets with U of T professors Hyun Kate Lee and Haley Wyatt in the department of biochemistry, in a process Mekhail likens to playing with toys. They ran the droplets through many tests, bouncing them against each other and observing their behaviour, which turned out to be very similar in a petri dish and in cells.

The most surprising finding came after several cycles of droplet fusion. “It was very bizarre and totally unexpected, I still remember the day,” Mekhail says. Oshidari observed that the larger droplets initiate an internal concentration of filament building blocks, forcing creation of a kind of self-interlocking brick road, which together with the spidery webs allow DNA to hook onto the longer autobahn filaments.

The complex process is easy to miss when looking at DNA damage sites largely because imaging in the field has become highly automated, Mekhail says, noting most software has been set up to see what has already been seen. “We can’t rely on the old ways of observing,” he says. “We need to update our software and also go back to looking with the human eye, guided by simulations when needed.”

The research was supported by the Canada Research Chairs Program, Canadian Institutes of Health Research, Ontario Ministry of Research and Innovation and the Natural Sciences and Engineering Research Council of Canada.

U of T yeast study prompts rethink of DNA replication and emergency repair

noreen.rasbach — Mon, 04 Feb 2019 05:00:00 +0000

U of T yeast study prompts rethink of DNA replication and emergency repair

noreen.rasbach Mon, 02/04/2019 - 00:00

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David Gallo (left) and his PhD adviser Grant Brown: Their findings are published in the journal Molecular Cell (photo by Jovana Drinjakovic)

Jovana Drinjakovic

Topic

Breaking Research

Donnelly Centre for Cellular & Biomolecular Research

Biochemistry

DNA

Graduate Students

Research & Innovation

Subheadline

DNA replication is more prone to errors at times of stress leading to mutations that could cause disease, research suggests

Cells go to great lengths to keep their genomes intact. But when resources are scarce, errors start creeping into the DNA code with potentially catastrophic consequences, new research suggests.

At no time is the DNA more vulnerable than when it is being copied during cell division so that it can be shared between descendent cells. To avoid mistakes during DNA replication, cells use proofreading enzymes that copy the code with high fidelity. Or so it was thought.

A new study led by Grant Brown, professor of biochemistry in the University of Toronto’s Donnelly Centre for Cellular and Biomolecular Research, suggests that at times of stress, DNA replication errors are far more frequent than previously appreciated. Although the researchers studied yeast cells, a similar process could ratchet up mutation rate in our own cells leading to cancer and other disease.

The findings have been described in the journal Molecular Cell.

The discovery took Brown and David Gallo, a PhD student who did most of the work, by surprise. They were studying DNA replication in cells raised under a limited supply of nucleotide bases that make up the A, T, C and G letters of the DNA code. “You can think of it as your car running out of fuel. We were removing gas from DNA replication,” explains Gallo.

Cells may encounter this type of stress when food is scarce or in disease, when resources are exhausted by fast-proliferating cancer cells, for example.

To make a copy of their genome, cells first unwind the double DNA helix into single strands, where each strand serves as a template against which new DNA is synthesized through complementary base pairing – A with T and C with G. This is usually done by DNA polymerase enzymes that are “super accurate and only make errors very rarely to ensure the blueprint of life is passed to the next generation with high fidelity,” says Brown.

But the lack of DNA building blocks led the cells to call on another type of DNA polymerase, which is more fallible. This was surprising because error-prone polymerases were thought to belong to an emergency repair machinery that is activated in response to physical damage, such as lesions in the DNA caused by UV light or some carcinogens. And while these enzymes act quickly to copy past the damaged part of the DNA, they also make mistakes.

This so-called “mutagenic repair” may seem an odd way to preserve the DNA, but it helps avoid worse genome-wrecking scenarios in which unwound DNA strands can lead to portions of chromosomes being lost.

“It’s better to copy and make some mistakes than to leave it uncopied and open to chromosomal rearrangements which would be much worse for the cell,” says Brown.

The study provides strong evidence of error-prone polymerases being used when there’s no apparent DNA damage. It suggests that these polymerases could be replicating more DNA than is appreciated and that this could be the source of more mutations that could lead to disease.

If the same is true for human cells, the finding could have implications for cancer research. Human cells have the same error-prone DNA copying machinery. And fast-proliferating cancer cells often experience what’s known as oncogene-induced replication stress, when they run out of fuel as DNA replication rates outstrip nucleotide supply. Under these conditions, the cells could resort to error-prone DNA replication, where new mutations could help cancer survive although this remains to be verified by future studies.

“The error-prone DNA replication pathway could very well be activated during oncogene induced replication stress to help cancer cells survive,” says Gallo. “This would make it a hot therapeutic target to selectively kill cancer cells.”

The study was supported by research grants from the Canadian Cancer Society, the Natural Science and Engineering Research Council of Canada and the Italian Association for Cancer Research.