This graphical abstract illustrates multiple phases of the DNA repair process carried out by high-resolution structures captured with cryogenic electron microscopy.
Illustration Credit: Charles Bell
Scientific Frontline: Extended "At a Glance" Summary: Structural Insights into DNA Repair Proteins and BRCA Mutations
The Core Concept: Researchers have captured the highest-resolution, multi-stage structural images to date of single-strand DNA annealing. By observing Mgm101—an ancestral yeast protein that serves as a model for the human DNA repair protein RAD52—scientists have mapped the precise physical phases of the DNA repair process.
Key Distinction/Mechanism: Previous imaging only captured the RAD52 protein bound to a single strand of DNA. Utilizing a combination of cryogenic electron microscopy (cryo-EM) and native mass spectrometry, this research successfully mapped multiple phases of the repair pathway. The mechanism involves the protein assembling into a 19-mer ring that acts as a template. It binds the first single strand of DNA by its sugar-phosphate backbone, leaving the nucleotide bases fully exposed in a newly observed "duplex intermediate" conformation, allowing it to efficiently search for and anneal with its complementary second strand before releasing the repaired double helix.
Major Frameworks/Components:
- RAD52 and Mgm101: Homologous proteins responsible for repairing broken DNA strands through a process called single-strand DNA annealing.
- 19-mer Molecular Complex: A large, multi-unit ring composed of 19 copies of the protein monomer, which functions as the structural template for DNA repair.
- Duplex Intermediate Phase: A previously unobserved conformation where the DNA backbone is bound to the protein ring, extending and unwinding the strand so complementary nucleotide bases can be matched.
- Cryogenic Electron Microscopy (Cryo-EM) & Mass Spectrometry: The advanced imaging and mass-measurement techniques required to capture the protein-DNA complexes across the substrate, intermediate, and product phases.
Branch of Science: Molecular Biology, Structural Biology, Biochemistry, and Oncology.
Future Application: The detailed structural snapshots will directly inform targeted drug development. Researchers plan to use this complete pathway map to design highly specific inhibitors capable of blocking the RAD52 repair mechanism in human cancer cells.
Why It Matters: Human cancer cells containing mutated BRCA1 and BRCA2 genes lack normal tumor-suppression functions and rely heavily on the RAD52 protein to repair their DNA, survive, and replicate. By fully understanding how this protein family binds and repairs DNA, pharmacologists can develop targeted therapies that disable RAD52, effectively killing cancer cells associated with breast, ovarian, and other BRCA-mutated cancers.
Scientists have captured the most detailed structural images to date of a specific type of protein’s DNA repair process, a finding that could reveal ways to inhibit the effects of BRCA1 and BRCA2 mutations that heighten the risk for breast, ovarian and other cancers.
Previous research has shown that a protein in humans called RAD52 performs DNA repair in cancer cells lacking the tumor-suppression function of normal BRCA genes, enabling the cells to survive and replicate – an indication that blocking RAD52 would kill these cells.
But blocking RAD52 requires fully understanding its repair activities, which have been difficult to capture with even the most sophisticated techniques. So the research team turned to its ancestral protein Mgm101 in yeast mitochondria and observed several key steps in its DNA repair process, called single-strand DNA annealing.
A clearer understanding of how this family of proteins binds to DNA strands and coaxes them back together after a break provides insights for drug targets that could halt the process in cancer cells empowered by mutated BRCA genes.
“It’s still a proposed mechanism: Just because we see these snapshots of the process doesn’t mean we know all the details, but we do have the best snapshots for any protein that does this single-strand annealing,” said senior author Charles Bell, professor of biological chemistry and pharmacology at The Ohio State University College of Medicine. “This focuses our strategies for drug development.”
DNA strands break every day in every cell, which is why proteins exist to fix the breaks and otherwise keep cellular processes running smoothly. But because repairs must happen quickly and human proteins are often more complex than their ancestral counterparts, even the most advanced imaging equipment can’t capture every step in the process.
Bell’s lab partnered on this research with the lab led by co-author Vicki Wysocki, professor emerita at Ohio State and chair of the School of Chemistry & Biochemistry at the Georgia Institute of Technology. Wysocki’s lab specializes in native mass spectrometry and mass photometry, using light to measure masses of protein-DNA complexes.
These techniques showed that Mgm101 assembled from a monomer, or single copy of itself, into a large multi-unit molecular complex called a 19-mer – essentially, a ring composed of 19 copies of the protein.
“This ring is sitting there as a template so that the first strand of the DNA can come down, and then the second strand comes on and starts being annealed to the first strand,” Wysocki said.
These findings were supported by what Bell’s lab determined using cryogenic electron microscopy, observing structures floating in solution and frozen in a thin layer of ice.
The high-resolution structures showed multiple phases of the process: the 19-mer ring with a single strand of DNA attached (substrate), with the second strand in place for annealing (duplex intermediate), and after release of the repaired DNA, visible as the classic double helix DNA formation (B-form product).
“RAD52 high-resolution structures have been determined with single-stranded DNA, but not with the two DNAs that it’s trying to anneal,” Bell said. “Its job is to bind single stranded DNA and anneal it to its complement sequence. It’s been captured structurally, but only in a few states relevant to the reaction.
“Here, we have more of the states along the full pathway from substrate, to intermediate and product. And the duplex intermediate is a conformation that’s never been seen before – when the protein binds the first DNA around the ring, it’s bound only by its sugar-phosphate backbone, with the nucleotide bases pointing up and fully exposed and separated, so that they can be searched. It’s extended, it’s completely unwound, and it’s circular.”
Bell said the field has been uncertain about whether this mechanism is carried out with one or two participating protein rings, but that these findings show the process is managed by a single molecular complex – and, therefore, single-strand annealing is likely to be a conserved cis mechanism.
The team plans to try to capture the same phases of the DNA repair process with RAD52 from humans, placing particular emphasis on the duplex intermediate, and to expand the role of mass spectrometry in determining how the DNA is bound to the protein.
Funding: This work was supported by the U.S. National Science Foundation and the National Institutes of Health. The cryo-EM data were collected at Ohio State’s Center for Electron Microscopy and Analysis and processed using the Ohio Supercomputer Center.
Published in journal: Nucleic Acids Research
Authors: Carter T Wheat, Zihao Qi, Miqdad Hussain, Katerina Zakharova, Vicki H Wysocki, and Charles E Bell
Source/Credit: Ohio State University | Emily Caldwell
Reference Number: mbio042726_01
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