Sunburns and aging skin are obvious effects of exposure to harmful UV rays, tobacco smoke and other carcinogens. But the effects aren’t just skin deep. Inside the body, DNA is literally being torn apart.
Understanding how the body heals and protects itself from DNA damage is vital for treating genetic disorders and life-threatening diseases such as cancer. But despite numerous studies and medical advances, much about the molecular mechanisms of DNA repair remains a mystery.
For the past several years, researchers at Georgia State University tapped into the Summit supercomputer at the Department of Energy’s Oak Ridge National Laboratory to study an elaborate molecular pathway called nucleotide excision repair, or NER. NER relies on an array of highly dynamic protein complexes to cut out, or excise, damaged DNA with surgical precision.
In their latest study, published in Nature Communications, the team built a computer model of a critical NER component called the pre-incision complex, or PInC. PInC plays a key role in regulating DNA repair processes in the latter stages of the NER pathway. Decoding NER’s sophisticated sequence of events and the role of PInC in the pathway could provide key insights into developing novel treatments and preventing conditions that lead to premature aging and certain types of cancer.
“We’re interested in the way cells repair their genetic material,” said lead investigator Ivaylo Ivanov, a chemistry professor at Georgia State University. “NER is a versatile pathway that repairs all kinds of different DNA damage using a three-stage process that relies on delicately balanced molecular machinery. Unfortunately, harmful mutations can develop that interfere with this machinery and cause severe human diseases.”
“Yet, the effects of genetic mutations can be strikingly different depending on their positions within the repair complexes. In some cases, mutations result in patients having UV light sensitivity and an extreme cancer predisposition. In other cases, they cause abnormal development and premature aging,” he said. “Why that happens is not completely understood at the molecular level. That’s the mystery our computer modeling efforts aim to unravel.”
The three acts of repair
NER unfolds in three distinct stages: recognition, verification and repair. Each stage requires different groups of proteins to perform specific functions, much like a trauma team has different specialists needed to treat injured patients in the emergency room. In that way, the NER machinery can adapt and change its shape depending on the task at hand.
In the first stage, the NER protein XPC (xeroderma pigmentosum group C) acts like a first responder that locates the site of the damaged DNA, or lesion, and then twists the DNA helix to make the damage accessible. XPC then calls in other repair proteins to help initiate the second stage, called damage verification, or lesion scanning.
Here, the NER protein machinery shifts into its next shape. As XPC steps back, the protein complex called transcription factor IIH, or TFIIH (pronounced T-F-2-H), moves into position. TFIIH further unwinds the section of DNA and scans the newly exposed strand for lesions.
After that, it’s in the hands of the surgeon — the PInC — in the third and final stage of repair.
With the “patient” stabilized and prepped for surgery, the operation to remove the damaged DNA strand can begin. Two enzymes, XPF and XPG (xeroderma pigmentosum groups F and G), position themselves precisely on each side of the lesion and act as molecular scissors to cut out the damaged segment of DNA.
Once the lesion is removed, new DNA is synthesized to fill in the gap left behind. Finally, the DNA backbone is sealed, and the damaged DNA is restored back to health.
“What we want to know is how the PInC forms after the lesion scanning phase,” Ivanov said. “How does it control the positioning of the two enzyme subunits that perform the dual incision of the damaged DNA strand? And importantly, is there any cross talk between the two enzymes? Do they sense each other?”
“That matters because once the damaged DNA strand is cleaved, it’s vital that the repair process is completed by filling in that gap,” he added. “Otherwise, it will lead to cell death or to the introduction of double-stranded breaks, which are extremely harmful to the cell.”
Answering those questions required the researchers to solve the structure of the PInC. In biology, understanding protein structure is essential for understanding the behavior or function of protein assemblies. The shapes, sizes and interactions of proteins determine how they fit together to form large biomolecular assemblies.
“We integrated the structural model of PInC using data from a variety of biophysical techniques, notably cryo-electron microscopy,” Ivanov said. “But in the end, the computation is what puts everything together.”
Much like the pieces of a jigsaw puzzle, the PInC model had to be assembled from known structures of constituent proteins, and all the individual pieces had to be put together in 3D. However, many of the PInC components had no known experimental structures.
To overcome this challenge, the researchers used a neural network-based model called AlphaFold2 to predict the unknown structures and the interfaces between the proteins that hold PInC together.
This Oak Ridge National Laboratory news article "Summit supercomputer draws molecular blueprint for repairing damaged DNA" was originally found on https://www.ornl.gov/news
