Time-resolved crystallography reveals the intricate steps of DNA repair by a photolyase enzyme
In a groundbreaking study, an international team of researchers has utilized time-resolved ultrafast crystallography to gain unprecedented insights into the process of DNA repair by a photolyase enzyme. This research marks the first-ever structural characterization of a complete enzyme reaction cycle, providing a deeper understanding of the intricate choreography between the enzyme and its substrate. Led by Manuel Maestre-Reyna, the study has received high praise from molecular biologist Aziz Sancar, who won the Nobel Prize in Chemistry in 2015 for his work on DNA repair mechanisms. This research has pushed the boundaries of time-resolved crystallography, capturing events that occur at vastly different timescales and mapping every enzymatic step of the process.
Photolyases and DNA Repair
Photolyases are enzymes that repair DNA damage caused by ultraviolet light in bacteria, fungi, plants, and certain animals. While humans and mammals do not possess these enzymes, they too experience light-induced damage. One common outcome of such damage is the formation of cyclobutane pyrimidine dimers (CPDs), which are responsible for skin cancer and sunburn. The photolyase enzyme repairs DNA by holding the CPD in its active site, where a coenzyme flavin adenine dinucleotide (FAD) transfers an electron to the cyclobutane ring, triggering a free-radical reaction that breaks the carbon-carbon bonds holding the pyrimidines together.
The Challenge of Capturing Enzymatic Steps
The study overcame the challenge of capturing events that occur at different timescales, allowing for the visualization of the entire DNA repair process. The initial electron transfer occurs after 100 picoseconds (ps), and the breaking of the second carbon-carbon bond takes place after approximately 1 nanosecond (ns). However, it takes about 500 ns for the enzyme’s active site to return to its initial state, followed by a further 200 microseconds (μs) for the repaired pyrimidines to flip out of the active site and the DNA to be released. Biophysicist Marius Schmidt highlights the difficulty of bringing together the fast events of bond formation and local relaxations with the slower catalytic cycles that complete on the millisecond timescale.
Time-Resolved Crystallography Reveals the Full Process
To capture the entire DNA repair process, Maestre-Reyna and his team utilized ultrafast crystallography on co-crystals of a microbial photolyase and CPD-containing DNA. They used two free electron laser (FEL) sources of bright x-rays, with one team collecting data for the first 10 ns of the reaction in Switzerland and the other team studying the relaxation of the enzyme complex and release of the DNA from 10 ns to 200 μs in Japan. The main challenge was the limited timeframe to collect the data, as photolyases are only active in their fully reduced form and experiments had to be performed under oxygen-free conditions. The researchers estimated they had approximately 20 hours before the enzyme became deactivated again.
A Multi-Disciplinary Team Effort
Maestre-Reyna emphasizes that the success of the study was made possible by a large multi-disciplinary team. Ming-Daw Tsai, Lars-Oliver Essen, Junpei Yamamoto, and Antoine Royant were key contributors to the research, each bringing their expertise in various fields such as structural basis of DNA repair, photolyase science, synthetic chemistry, and crystal spectroscopy, respectively. The collaboration and shared knowledge were crucial in unraveling the complex DNA repair process.
Uncovering the Role of Water Molecules
One significant yet poorly understood aspect of the reaction is the presence of a cluster of five water molecules within the enzyme’s active site. This cluster, hydrogen-bonded to a part of the protein, appears to fine-tune the active site’s affinity for CPDs and enables quick reorganization when the initial electron transfer occurs. The study confirms a previously speculative idea and sheds light on the role of water molecules in the DNA repair process.
Conclusion:
The groundbreaking research utilizing time-resolved ultrafast crystallography has provided a comprehensive understanding of the DNA repair process by a photolyase enzyme. By visualizing the intricate steps and choreography between the enzyme and its substrate, this study has pushed the boundaries of time-resolved crystallography. The findings not only enhance our knowledge of DNA repair mechanisms but also pave the way for the development of new strategies to combat DNA damage-related diseases. The collaborative effort of the multi-disciplinary team highlights the power of collective expertise in unraveling complex biological processes. As further studies build upon these findings, the potential for breakthroughs in DNA repair research becomes increasingly promising.
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