Fixing gene breakages quickly and perfectly can be a matter of life or death for most organisms. Even the simplest changes in a sequence risk a catastrophe, especially if the altered code is responsible for a critical function.
Over the past half century, biologists have studied the mechanisms involved in combining most of the major steps to making DNA-faithful repairs. Still, part of the process has been frustratingly unclear.
Marking key enzymes and DNA with fluorescent tags and observing the repair process in real time in a Escherichia coli model, researchers at Uppsala University in Sweden have filled in missing details about how bacteria find the templates they rely on to keep error-free genetic repairs.
One trick that most living things use to keep their code in order is the process of homologous recombination, the biological equivalent of comparing two different versions of a script to make sure a copy has not been entered error by mistake.
By maintaining an undamaged version of a sequence next to a repair job, a cell can ensure that no changes occur when the cut ends are stuck.
Molecular biologists have known for some time that the RecA recombinase protein plays a key role in the management of this process. It is such an important enzyme to maintain the integrity of DNA that some version of it has been found in virtually all species studied.
When a double-stranded DNA “scale” is fully installed, a protein complex is put to work by grabbing the cut ends and trimming it neatly so that RecA can settle in and do its job.
This implies that the protein stretches into a long cluster, forming a protein and nucleic acid filament capable of being contained in both the broken strand and an intact second scale of unbroken DNA.
Scientists know this very well. From here, the filament must find the right sequence to serve as a point of comparison. How the filament handles this search in a fairly short time has been a mystery for most of the 50 years, one that has been added to the millions of base pairs that should be checked amid the complex twists and turns of the chromosome.
To better understand the time and navigation of the enzyme at work, the researchers grew thousands of people E. coli cells within a series of tiny channels that allowed them to track individual bacteria as they experimented.
With the cells in place, the scientists made precise pauses in their DNA by editing the CRISPR gene, marking the cut-off ends with fluorescent markers to visualize the location of the break under the microscope.
“The microfluidic culture chip allows us to track the fate of thousands of bacteria simultaneously and monitor CRISPR-induced DNA ruptures in time,” says Jakub Wiktor, a molecular biologist at Uppsala University.
Finally, they used antibodies to identify the location of the RecA filaments as they were installed and searched the library.
A chemical alert informed the team when the entire repair process was complete. On average, it took only 15 minutes to do so E. coli to finish the job.
Surprisingly, it usually took the protein nine of those minutes to find the right template.
The secret seems to lie in the construction of the RecA nucleoprotein filament. This thread stretches across the cell, grabbing the chromosome and sliding down in search of a match with the sequence at its disposal.
While this may not seem so efficient, it’s really no different than methodically walking up and down the aisles of a library in search of a book that matches the call number in the catalog.
“Because the ends of DNA are incorporated into this fiber, it is enough for any part of the strand to find the beautiful template, and therefore the search is theoretically reduced from three to two dimensions,” says Arvid Gynnå.
“Our model suggests that this is the key to a quick and successful homology repair.”
Although this research was conducted on bacteria, the fact that RecA is so similar to the entire biosphere makes it relevant to our own bodies.
Now that we know how the process works, we can begin to look for signs of situations where repairing our own DNA goes wrong, paving the way for understanding the origins of diseases like cancer.
This research was published in Nature.