Digging into DNA repair with Optical Tweezer technology

By combining an optical tweezer technology called C-trap that manipulates a single DNA molecule and a new approach, researchers were able to get a detailed view of how cells find and repair damaged DNA.

Their findings are described in an article titled “Single molecule DNA-binding protein analysis from nuclear extracts (SMADNE),” Posted in Nucleic acid research.

In the new study, the researchers used the C-trap to investigate how different DNA repair proteins identify and bind to their respective forms of damage.

“I like to think of DNA damage as a pothole,” explained Ben Van Houten, PhD, a professor in Pitt’s Department of Pharmacology and Chemical Biology. “In a particular DNA repair pathway, it takes about 30 proteins to go from finding the pothole to putting the repair patch in place. Although we can’t observe all of these proteins at the times, we can observe them two by two.

Van Houten’s lab is interested in repair proteins that repair DNA damage caused by environmental factors, such as the sun’s ultraviolet (UV) rays and environmental pollutants. The C-trap system is based on a Nobel Prize-winning technology called optical tweezers, which uses a powerful beam of light to grab and move microscopic beads until they stick to either side of a molecule. .

“You can move the two beads together and hope that the two ends of the DNA grip each bead like Velcro. When you move the beads apart, you can actually feel the measurement of the force of the DNA like a spring. or a rubber band,” said first author Matthew Schaich, PhD, postdoctoral fellow in the Van Houten lab.

In collaboration with researchers at the University of Kent, the Van Houten group has developed a new method called single molecule analysis of DNA-binding proteins from nuclear extracts, or SMADNE.

Using SMADNE, the researchers extracted DNA repair proteins from the cell nucleus. They then introduced these proteins into the C-trap and analyzed how and when they bound to DNA containing various types of damage.

“You have an area of ​​DNA damage and you want to know how a cell can identify and repair it,” Schaich explained. “One of the most important things to understand is which arrives first. Once it arrives, does it stay through the entire repair cascade? Does it transfer the repair to another protein? With the C -trap, one can watch the comings and goings of proteins and learn a lot about assembly and disassembly orders.

Van Houten thinks of DNA repair proteins like people socializing in a bar.

“Two people walk into a bar. Who walks through the door first? How long do they sit together at the bar, then who leaves the bar first? DNA repair proteins, like people, are dynamic,” Van Houten said.

The researchers found that when DDB1 and DDB2 worked together at the damaged site, they usually arrived at the DNA together and left together, as expected. But to their surprise, they also observed 11 different association and dissociation patterns, with the two proteins arriving and leaving at different times.

In addition to DDB1 and DDB2, Van Houten’s team used C-trap and SMADNE to study DNA repair protein activities of several different repair pathways to better understand these repair systems.

Their findings may pave the way for a better understanding of how disruptions in these pathways can lead to diseases such as cancer, and may pave the way for new treatments.

“DNA repair is a double-edged sword,” Van Houten explained. “If you don’t have an effective repair, environmental stressors could cause enough damage for cancer to grow. On the other hand, many cancer treatments kill tumors by targeting DNA repair mechanisms.

Van Houten and his team have filed a patent application for their SMADNE system and will continue to analyze the 30 proteins of this UV damage repair pathway.

“The combination of C-trap and SMADNE has opened up endless possibilities for studying DNA repair. But what is the most important question we can answer using this new tool? says Van Houten. “For me, it’s about knowing the precise role of each of the proteins in this pathway.”