The DNA in every cell in your body undergoes double-strand breaks multiple times a day. Survival requires a repair mechanism for this. Over billions of years, we have evolved to be very good – but not perfect – at fixing these mistakes as they occur. When errors are not corrected, cancer can be the consequence, so understanding how the repair mechanism operates could be essential to treating disease. Dr Donna Whelan of Australia’s La Trobe University is not the first to witness the DNA repair process, but earlier efforts have often involved rather artificial conditions. For example, the damage proteins were fixing in previous studies were often those induced by shining powerful laser light on the cells or exposing them to damaging chemicals. Instead, Whelan has used a process known as multicolor single-molecule localization microscopy to observe “first responder” proteins as they rush to repair the ordinary DNA breaks that occur even under non-stressful conditions. Single-molecule localization microscopy was initially developed for use by physical chemists, who Whelan told IFLScience, were doing “science for science's sake”, rather than having clear applications in mind. Transferring this to functioning cells took effort. “Some of the first images within cells took 12 hours,” Whelan said. Naturally at that rate it there was no opportunity to watch processes evolve that take seconds or minutes. However, over time Whelan said; “The technology has been streamlined and optimized.” The consequence is Whelan has she has something of the order of 10,000 images for her paper in Proceedings of the National Academy of Sciences describing the work, revealing the process in stunning detail. (Left) A cancer cell as seen using single-molecule localization microscopy, with nDNA in magenta, MRE11 in blue, and CtIP in yellow. (Right) How conventional microscopy produces a blurry, mostly unhelpful image, whereas the single-molecule approach builds up the image by pinpointing the locations of the molecules themselves to create something much clearer. The most important thing Whelan has learned from her observations, she told IFLScience, is the amount of redundancy in our repair mechanisms. In a paper published as the technique was still developing, she reported “a protein no one thought was important” appears to act as a backup for the BRCA2 protein. BRCA2 is famous for its association with breast cancer, but that is a consequence of rare mutations in the gene that codes for it. When functioning normally, BRCA2 plays an essential part in cell repair. The discovery of a protein whose activity might be stimulated to upgrade its substitute role has, Whelan noted; “Huge therapeutic potential.” There are no similarly immediate implications from Whelan's latest publication, but the demonstration of the extraordinary capacity of single-molecule localization microscopy certainly looks like the beginnings of something exciting. The paper's major focus was on the fact the body has two forms of cell repair. A quick-and-dirty approach has proteins grabbing the ends of DNA and sticking them back together, “hoping there will be no mutations,” as Whelan puts it. The more sophisticated version, known as Homologous Recombination (HR), involves searching for a similar sequence to act as a template. This is slower but produces fewer errors. The process by which cells select which to use when a double strand-break occurs is poorly understood, and Whelan thinks her work has provided a starting point to improve that. The protein 53BP1 is “known to antagonize HR,” the paper notes. While this may sound familiar to those who have worked for large companies, Whelan showed 53BP1 never actually gets right to the site of the repair activity, instead being recruited to the vicinity early on, playing some important role on the sidelines. Source
