Researchers achieve breakthrough in understanding DNA repair through ultrafast crystallography
In a groundbreaking study, an international team of researchers has utilized time-resolved ultrafast crystallography to track the intricate process of DNA repair by a photolyase enzyme. This research marks a significant milestone as it represents the first comprehensive structural characterization of a complete enzyme reaction cycle. Led by Manuel Maestre-Reyna, the team’s work offers unprecedented insights into the choreography of both the enzyme and its substrate, providing a deeper understanding of the DNA repair process.
Unveiling the Dance of DNA Repair:
To comprehend the significance of this research, it is essential to grasp the process of DNA repair. Photolyases are enzymes that repair DNA damage caused by ultraviolet light in various organisms, including bacteria, fungi, plants, and certain animals. While humans lack these enzymes, we also experience light-induced damage, such as the formation of cyclobutane pyrimidine dimers (CPDs). These CPDs, responsible for skin cancer and sunburns, occur when adjacent pyrimidine bases fuse together via a four-membered cyclobutane ring.
The DNA repair process involves the photolyase enzyme binding to the CPD in its active site. A coenzyme called flavin adenine dinucleotide (FAD) transfers an electron to the cyclobutane ring, initiating a light-stimulated free-radical reaction that breaks the carbon-carbon bonds holding the pyrimidines together. While the initial electron transfer and bond breakage occur rapidly (within 100 picoseconds and 1 nanosecond, respectively), the enzyme’s active site takes approximately 500 nanoseconds to return to its initial state. Additionally, it takes an additional 200 microseconds for the repaired pyrimidines to flip out of the active site and the DNA to be released.
The Challenge of Time-Scale Integration:
The research conducted by Maestre-Reyna and his team tackles the challenge of capturing events that occur at vastly different timescales, allowing for the mapping of every enzymatic step in the DNA repair process. Biophysicist Marius Schmidt describes this challenge, stating that while enzymes complete their catalytic cycles in milliseconds, the fundamental events like bond formation and local relaxations happen at an extremely fast pace. Bridging these two timescales has proven to be a formidable task.
The Breakthrough: Ultrafast Crystallography:
To overcome this challenge, the researchers employed ultrafast crystallography on co-crystals of a microbial photolyase and CPD-containing DNA. They utilized two free electron laser (FEL) sources of bright x-rays, with one team collecting data for the first 10 nanoseconds of the reaction and the other team studying the relaxation of the enzyme complex and the release of DNA from 10 nanoseconds to 200 microseconds.
The main hurdle in this research was the need for rapid data collection. Photolyases are only active in their fully reduced form, requiring all experiments to be conducted under oxygen-free conditions. Given the enzyme’s susceptibility to oxidation, the researchers estimated that they had a mere 20-hour window before deactivation. Within this time frame, they successfully purified the protein, activated it, co-crystallized it, harvested the crystals, and collected the data on-site.
The Collaborative Effort:
Maestre-Reyna emphasizes that this groundbreaking work was only possible due to the collaboration of a large multi-disciplinary team. The team included experts such as Ming-Daw Tsai, who has extensively studied the structural basis of DNA repair, Lars-Oliver Essen, a renowned photolyase scientist, Junpei Yamamoto, a synthetic chemist capable of producing photodamaged DNA at the required scale, and Antoine Royant, an expert in performing spectroscopy in crystals.
Unraveling the Mysteries of DNA Repair:
The research not only sheds light on the intricate dance of DNA repair but also unravels previously little-understood aspects of the process. One such component is a cluster of five water molecules within the enzyme’s active site. This cluster, hydrogen-bonded to a specific part of the protein, fine-tunes the active site’s affinity for CPDs and enables rapid reorganization when the initial electron transfer occurs. This discovery confirms a previously speculative idea and provides further insights into the DNA repair process.
Conclusion:
The breakthrough achieved by Maestre-Reyna and his team through time-resolved ultrafast crystallography opens the door to a deeper understanding of DNA repair. By visualizing the complete enzyme reaction cycle, researchers have gained unprecedented insights into the choreography of both the enzyme and its substrate. This research not only advances our knowledge of DNA repair but also paves the way for the development of potential therapeutic interventions for conditions related to DNA damage, such as skin cancer. As the dance of DNA repair becomes clearer, the potential for harnessing its intricacies for the benefit of human health becomes ever more promising.
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