Occasionally, the overall “fit and finish” of DNA is marred by a bubble—a region where complementary DNA strands aren’t complementary at all on account of a sequence of unpaired bases. When a bubble occurs, a protein called XPG rushes to the scene and instigates a repair. But XPG isn’t, as you might think, all about smoothing and polishing. Instead, XPG begins by seemingly making matters worse.
XPG binds bubble DNA and breaks the stacking of unpaired bases, bending DNA severely. Only then does XPG recruit and load into place other proteins that finally smooth things out.
This repair mechanism was uncovered by scientists working at Lawrence Berkeley National Laboratory (Berkeley Lab). Combining data from crystallography, biochemistry, small angle X-ray scattering (SAXS), and electron microscopy, these scientists detailed how XPG works at the molecular level—which is no mean feat, considering that XPG largely consists disordered elements.
Detailed findings appeared this month in the Proceedings of the National Academy of Sciences, in an article titled, “Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations.” As this article’s title suggests, the new XPG findings not only describe how DNA repair works in healthy cells, they also suggest how different mutations can translate into different diseases and cancer.
“[Our data] unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both nucleotide excision repair bubble junctions and replication forks,” the article’s authors wrote. “Collective results support XPG scaffolding and DNA sculpting functions in multiple DNA damage response processes to maintain genome stability.”
“Our analysis,” the authors added, “[also provides] predictions of the structural impacts of XPG disease mutations associated with two phenotypically distinct diseases: xeroderma pigmentosum (XP, skin cancer prone) or Cockayne syndrome (XP/CS, severe progressive developmental defects).”
Although the extent of what XPG does in human cells is still only partially understood, the protein has long been considered essential to human health. When XPG is missing or fails to function normally, devastating diseases occur. One such disease is xeroderma pigmentosum, a condition of varying severity characterized by extreme sun sensitivity and greatly elevated risk of skin cancer. Another is Cockayne syndrome, which is characterized by a progressive and ultimately fatal neurological decline that begins in infancy. Both xeroderma pigmentosum and Cockayne syndrome are both known to be caused by mutations in the gene that encodes XPG.
Fascinated by XPG’s many roles, Berkeley Lab scientists Susan Tsutakawa and Priscilla Cooper, along with John Tainer, the director of structural biology at the University of Texas MD Anderson Cancer Center and a visiting faculty member at the Berkeley Lab, 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.
Previously, the scientists had discovered that XPG often binds to damaged DNA without engaging its DNA cutting activity, but they 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, multi-unit molecule’s overall structure using a trifecta of cutting-edge imaging technology.
They performed X-ray crystallography at Stanford Synchrotron Radiation Laboratory, and 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 that have 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 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.
“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 Tsutakawa, the first author of the current work. “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.”
“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,” added Cooper, a study co-author. “XPG then uses its sculpting properties to bend the DNA to recruit and load into place the proteins that can fix that type of damage.”
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.