This enigmatic protein sculpts DNA to repair harmful damage

June 22, 2020

Sometimes, when something is broken, the first step to fixing it is to break it even more.

In a recent example, scientists seeking to understand the mechanism of a DNA-repairing enzyme have discovered that the molecule performs its functions by first marking and then further breaking damaged DNA. The team's surprising findings on the protein, called XPG, have provided much-needed insight into how DNA repair works in healthy cells, as well as how different mutations can translate into different diseases and cancer.

"We saw that XPG makes a beeline for discontinuous DNA - places where the hydrogen bonds between bases on each strand of the helix have been disrupted - and then it very dramatically bends the strand at that exact location, breaking the interface that connects bases stacked on top of each other," said Susan Tsutakawa, a structural biologist in the Biosciences Area at Lawrence Berkeley National Laboratory (Berkeley Lab) and first author on the work, published this month in PNAS. "The bending activity adds to an already impressive arsenal, as XPG was first identified as a DNA chopping enzyme, responsible for cutting out nucleotide bases with chemical and UV radiation damage."

Yet despite this knack for destruction, the team notes that XPG is more like a master sculptor than a demolition crew.

"An unexpected finding from our imaging data is that the flexible parts of the protein - which were previously impossible to examine - have the ability to recognize perturbations associated with many different types of DNA damage," said co-author Priscilla Cooper, a biochemist senior scientist in the Biosciences Area. "XPG then uses its sculpting properties to bend the DNA in order to recruit and load into place the proteins that can fix that type of damage."

A protein with many jobs

Although the extent of what XPG does in human cells is still only partially understood, scientists have long known that the protein is essential to human health by observing the devastating symptoms that occur when it is missing or not functioning normally. Cockayne syndrome, a disease characterized by a progressive and ultimately fatal neurological decline that begins in infancy, and xeroderma pigmentosum, a condition of varying severity characterized by extreme sun sensitivity and greatly elevated risk of skin cancer, are both known to be caused by mutations in the gene that encodes XPG.

Fascinated by its many roles, Tsutakawa, Cooper, and John Tainer, the director of structural biology at the University of Texas MD Anderson Cancer Center and visiting faculty in the Biosciences Area, have been collaborating on studies of XPG for 20 years. The trio, and their many colleagues, pool their expertise in structural biology, molecular imaging, biochemistry, and cell biology so that they can map the protein's structure and interpret how its three-dimensional form interacts with DNA and other proteins. They had previously discovered that XPG often binds to damaged DNA without engaging its DNA cutting activity, but could not examine the protein in great enough detail to find out what it actually does in these instances.

After many years spent developing technology that could catch up with their ambitions, the team was finally able to build a precise model of XPG's catalytic core - the region responsible for the DNA cutting activity - and produce images of the large, multiple-unit molecule's overall structure using a trifecta of cutting-edge imaging technology.

They performed X-ray crystallography at Stanford Synchrotron Radiation Laboratory, and small angle X-ray scattering (SAXS) at the SIBYLS beamline of Berkeley Lab's Advanced Light Source. SAXS is a technique that has recently evolved to allow scientists to analyze flexible molecules moving freely between their natural states rather than in static or frozen conformations, as necessitated by crystallography. Such an approach is sorely needed for a protein like XPG, whose catalytic core is only one-quarter of the total structure and the rest is made of highly flexible "disordered" regions with no default shape.

To visualize the XPG-bound DNA, the scientists recruited Jack Griffith, a pioneer of rotary shadowing electron microscopy at the Lineberger Comprehensive Cancer Center at UNC Chapel Hill. Rotary shadowing electron microscopy allows direct visualization of individual DNA molecules with proteins bound to them, including how they were bent by XPG.

"The ability to see the shapes of individual DNA molecules gave us an essential clue as to how XPG works to identify and process damaged DNA," said Griffith, a professor of biochemistry and biophysics and expert in protein-DNA interactions.

The electron microscopy imaging also provided visual evidence supporting the scientists' previous surprising finding that XPG plays a role in homologous recombination - a DNA repair process frequently used by cells to fix dangerous double-strand breaks before replication. This means that XPG could be at the right place to help known homologous recombination proteins such as BRCA1 and BRCA2, defects in which are known to cause cancer.

Meanwhile, crystallography performed on the catalytic core shed light on how inherited patient mutations in the gene for XPG can translate into severe protein dysfunction and different diseases. The team made and tested catalytic core proteins having each of the 15 known point mutations that cause either xeroderma pigmentosum or Cockayne syndrome, and found that these single amino acid substitutions can destabilize the entire protein, but to different extents. The properties of the residual mutant protein will determine which disease results. "This structure helps us understand the distinction between the two diseases," said Cooper, "and it reinforces how complex the protein is."

Invigorated by the new information, the team has already begun a study looking at XPG's role in different cancers, as well as a follow-up structural study of the protein's disordered regions to learn more about its DNA sculpting properties.

"The superb technical and collaborative strengths of Berkeley Lab and our partners made this multi-disciplinary breakthrough feasible," noted Tainer.

"But we would also like to highlight the contribution of patients and patients' families," added Tsutakawa. "So much of what we have discovered was made possible by them choosing to share their DNA sequences with the scientific community."
-end-
The Advanced Light Source (ALS) and Stanford Synchrotron Radiation Laboratory are Department of Energy (DOE) Office of Science user facilities. The SIBYLS beamline 12.3.1 at the ALS is also funded by the NCI Structural Biology of DNA Repair Program and the NIH through ALS-ENABLE.

Founded in 1931 on the belief that the biggest scientific challenges are best addressed by teams, Lawrence Berkeley National Laboratory and its scientists have been recognized with 13 Nobel Prizes. Today, Berkeley Lab researchers develop sustainable energy and environmental solutions, create useful new materials, advance the frontiers of computing, and probe the mysteries of life, matter, and the universe. Scientists from around the world rely on the Lab's facilities for their own discovery science. Berkeley Lab is a multiprogram national laboratory, managed by the University of California for the U.S. Department of Energy's Office of Science.

DOE's Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit energy.gov/science.

DOE/Lawrence Berkeley National Laboratory

Related Cancer Articles from Brightsurf:

New blood cancer treatment works by selectively interfering with cancer cell signalling
University of Alberta scientists have identified the mechanism of action behind a new type of precision cancer drug for blood cancers that is set for human trials, according to research published in Nature Communications.

UCI researchers uncover cancer cell vulnerabilities; may lead to better cancer therapies
A new University of California, Irvine-led study reveals a protein responsible for genetic changes resulting in a variety of cancers, may also be the key to more effective, targeted cancer therapy.

Breast cancer treatment costs highest among young women with metastic cancer
In a fight for their lives, young women, age 18-44, spend double the amount of older women to survive metastatic breast cancer, according to a large statewide study by the University of North Carolina at Chapel Hill.

Cancer mortality continues steady decline, driven by progress against lung cancer
The cancer death rate declined by 29% from 1991 to 2017, including a 2.2% drop from 2016 to 2017, the largest single-year drop in cancer mortality ever reported.

Stress in cervical cancer patients associated with higher risk of cancer-specific mortality
Psychological stress was associated with a higher risk of cancer-specific mortality in women diagnosed with cervical cancer.

Cancer-sniffing dogs 97% accurate in identifying lung cancer, according to study in JAOA
The next step will be to further fractionate the samples based on chemical and physical properties, presenting them back to the dogs until the specific biomarkers for each cancer are identified.

Moffitt Cancer Center researchers identify one way T cell function may fail in cancer
Moffitt Cancer Center researchers have discovered a mechanism by which one type of immune cell, CD8+ T cells, can become dysfunctional, impeding its ability to seek and kill cancer cells.

More cancer survivors, fewer cancer specialists point to challenge in meeting care needs
An aging population, a growing number of cancer survivors, and a projected shortage of cancer care providers will result in a challenge in delivering the care for cancer survivors in the United States if systemic changes are not made.

New cancer vaccine platform a potential tool for efficacious targeted cancer therapy
Researchers at the University of Helsinki have discovered a solution in the form of a cancer vaccine platform for improving the efficacy of oncolytic viruses used in cancer treatment.

American Cancer Society outlines blueprint for cancer control in the 21st century
The American Cancer Society is outlining its vision for cancer control in the decades ahead in a series of articles that forms the basis of a national cancer control plan.

Read More: Cancer News and Cancer Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.