Dynamic single-molecule offers a direct view of protein interactions in real time. Credit: Artur Plawgo/ Getty Images
Guarding genes against damage is a crucial and complex job, so it’s to be expected that cells have several mechanisms to repair DNA breakages, each providing support under different circumstances.
For many of these pathways, scientists have long known the proteins involved. This knowledge came primarily from traditional biochemical assays, which identify the molecules that bind to DNA during repair, but cannot explain the intermediate and dynamic protein activity — often missed because of the heterogeneous assay outputs. It’s like knowing the ingredients for a dish but having no idea how they combine to produce the end result.
“Observing the dynamics of DNA-interacting proteins, rather than their static structure, can generate ground-breaking mechanistic insights,” says Andrea Candelli, chief scientific officer of life sciences company, LUMICKS.
The reward for unlocking the mysteries of DNA repair could be significant. For instance, women with defective versions of the BRCA repair proteins have a much higher risk of aggressive breast and ovarian cancers. These cancers instead rely on the PARP repair pathway, making this back-up pathway a solid target for anti-cancer treatments.
The key to building on this success for a wider range of cancers is understanding the delicate, fleeting interactions between proteins and DNA that drive complex repair processes. “The ability to visualize enzymes at a single molecule level has been transformative,” says geneticist, Simon Boulton from the Francis Crick Institute in London. He uses dynamic single-molecule imaging, which offers a chance to see activity in real time and get direct proof of the mechanisms at work.
“Single-molecule techniques deepen our understanding of disease through these mechanisms, which could yield new insights into cancer development and identify new targets for treatment," Candelli says.
Solving a 30-year mystery
Boulton became interested in dynamic single-molecule analysis when studying RAD51 — an enzyme regulated by BRCA2 to repair double-strand DNA breaks. Multiple RAD51 proteins bind along the length of broken single-stranded DNA to form filaments, which allows the damaged strands to be restored by lining up with their complete strands. Although RAD51 and similar proteins had been implicated in this process for three decades, how the filaments formed was a mystery.
“We essentially hit an impasse in understanding detailed mechanisms because we were relying on biochemical assays, which just give you an endpoint. They don’t really tell you about the intermediate stages and the dynamics of the system,” says Boulton.
The C-Trap dynamic single-molecule imaging system, produced by LUMICKS, allowed his team to see the activity in real time. The C-Trap uses optical tweezers to hold beads attached at either end of DNA strands, creating a rope bridge-like structure. With the DNA pinned in place, it is straightforward to trace the motion of fluorescently-labelled proteins under a microscope and view their activity.
This imaging showed that smaller proteins bind to the end of the filament — to stabilize the RAD51’s DNA binding, before detaching and repeating the process further along. This, in turn, allows the filament to grow1. “We would never have known that if we hadn’t seen these proteins act in real time at a single molecule level,” says Boulton. “This work was transformative: it explained findings from the last 30 years. Finally, we were able to nail how this complex works.”
What makes this imaging tool so unique?
Award-winning tech
The C-Trap system takes advantage of the power of optical tweezers, a biophysical technique that uses laser beams to control the movement of microscopic objects. This light-trapping approach was recognized by the 2018 Nobel Prize in Physics, although its adoption in molecular biology remained limited.
“Optical tweezers were very much in the realm of hardcore biophysics until recently,” says Boulton. But, even as a geneticist, he has found the C-Trap set-up easy to use: a unique yet simple tool. “It’s been designed so people who aren’t conventional biophysicists can quickly learn to use the system and start their experiments.”
The system is not limited to observing proteins at work. The versatility of the optical tweezers means that researchers can also manipulate and measure the DNA’s structure. When the DNA strands were stretched by the tweezers, Boulton could see the tension drop after RAD51 was added. As the protein helped to unwind the helix and make it longer, it resulted in the observed decreased force. Other researchers have used this method to understand how the accuracy of CRISPR/Cas9 gene editing changes as DNA is distorted under strain2. These insights could help to improve the process for more accurate editing in vivo.
“Nobody had seen that before”
Following success with RAD51, Boulton applied the same approach to another important DNA repair protein, HELQ. His lab’s previous work showed which other molecules HELQ interacts with, but not how. With the C-Trap, he could see individual molecules of HELQ travelling along single DNA strands, unwinding them from their usual helix shape. Surprisingly, HELQ travels much faster along the DNA when it forms a complex with RAD51, raising new questions about this process and bringing previously unknown functions to light3.
It also revealed that HELQ has another role: it can zip up two aligning strands of DNA to form the double helix. HELQ tethers to proteins that cover the single-stranded DNA, and then strips away this layer to facilitate the joining of complementary strands. This process is an alternative to the BRCA2-driven mechanism for repairing double-strand breaks in DNA and could be a useful target for new cancer therapies.
“It was really cool — nobody had seen that before,” says Boulton. “We could then show in cells that this activity is important in several DNA repair processes.”
Future insights
Analysing one molecule at a time provides a useful viewpoint into how proteins interact with DNA. Boulton is convinced that it represents an indispensable investment: “It’s definitely worth the time commitment to gain those dynamic insights.”
The C-Trap approach even works on one of the toughest targets — the BRCA2 protein itself. It is about 10 times the size of RAD51, which makes it difficult to work with. Purified BRCA2 has been available only since 2010, so the subject of just over a decade of biochemical investigation4.But now, Boulton can look at individual molecules, such as RAD51 or HELQ, interacting with DNA. “It’s a miracle we’ve been able to do this,” he says.
While this work is still preliminary, Boulton is clear that dynamic single-molecule imaging is leading to significant, previously unreachable discoveries. He hopes to unravel many more mysteries of the enzymes involved in DNA repair and beyond. “We’ve gained some interesting and unexpected insights into how BRCA2 behaves. That only came from direct visualization.”