Clear picture of how the “first responder” is recruited to DNA damage sites

DNA is often likened to a blueprint. The particular sequence of As, Cs, Gs and Ts in DNA provides information for building an organism.

What is not captured by this analogy is the fact that our DNA requires constant maintenance to maintain its integrity. If there were no dedicated DNA repair machines that regularly correct errors, the information contained in DNA would be quickly degraded.

This repair occurs at cell cycle checkpoints that are activated in response to DNA damage. Like a quality assurance agent on an assembly line, the proteins that participate in the DNA damage checkpoint evaluate the cell’s DNA for errors and, if necessary, suspend cell division and carry out repairs. When this checkpoint breaks down – which can happen as a result of genetic mutations – DNA damage accumulates and the result is often cancer.

Although scientists have learned a great deal about DNA damage and repair over the past 50 years, important questions remain unanswered. A particularly troubling puzzle is how a repair protein called 9-1-1 clamp – a “first responder” to DNA damage – attaches to the site of a broken DNA strand to activate the stitch. DNA damage control.

“We know that this attachment is a critical step needed to initiate an effective repair program,” says Dirk Remus, a molecular biologist at the Sloan Kettering Institute (SKI) who studies the fundamentals of DNA replication and repair. “But the mechanisms involved are completely obscure.”

Now, thanks to a collaboration between the lab of Dr. Remus and that of SKI structural biologist Richard Hite, a clear picture of how the 9-1-1 clamp is recruited to sites of DNA damage has emerged. . The results, which challenge conventional wisdom in the field, were published on March 21, 2022 in the journal Structural and Molecular Biology of Nature.

Complementary expertise yields surprising results

The surprising discoveries were born from a collaboration between two laboratories with complementary skills. Dr. Remus’ lab uses biochemical methods to study the process of DNA replication and repair. One of the main goals of his research over the past few years has been to reconstruct the entire process of DNA replication and repair in a test tube, except for a surrounding cell.

As a result of this effort, his lab has purified several components of the repair machinery, including 9-1-1 proteins and proteins that facilitate binding of 9-1-1 to DNA.

Dr. Remus realized that if these complexes could be visualized at atomic resolution, they would provide a set of freeze-frame images of individual steps in the repair process. It was then that he turned to Dr. Hite’s lab for help.

“I said, ‘We have this complex; can you help us determine its molecular structure to understand how it works? And that’s what he did. »

Dr. Hite is a structural biologist specializing in the use of a technique called cryo-electron microscopy (cryo-EM), which allows the study of proteins and protein assemblies by visualizing their fine-grain motions at resolutions that can reveal the positions of individual amino acids. acids in proteins. Like the gears and levers of a machine, it is these movements of amino acids that enable proteins to serve as the workhorses of the cell, including those that repair DNA.

“When Dirk came to us, we realized that many of the tools our lab had developed over the past few years were perfectly suited to answering this question,” says Dr. Hite. “Thanks to cryo-EM, we are able to determine not only a structure, but a set of structures. By putting these structures together in a logical diagram, based on the new data and the previous biochemical data, we can propose a proposal on the functioning of this clamp.

They did, and the results were amazing.

“The model we developed had some interesting features that contradicted what had previously been thought to be how these types of clamps are loaded onto DNA,” says Dr Hite.

A new model for opening and closing a DNA clamp around DNA

The 9-1-1 clip is shaped like a ring. To perform its function, it must surround the broken DNA at the junction between an exposed end of a strand of a piece of double-stranded DNA adjoining a single strand. Therefore, the ring structure of the 9-1-1 clamp must open to allow single-stranded DNA to dangle in the center of the clamp and then close around it. This does not occur spontaneously but is facilitated by another protein complex, called the clamp loader complex.

“Based on all previous studies, it had been thought that the clamps would open like a lock washer, where essentially the two open ends of the clamp would rotate out of plane to create a narrow gap,” says the Dr Remus. “But what Rich has observed is that the 9-1-1 clamp opens much wider than expected, and it opens completely in plane – there’s no twist like in the lock puck scenario.”

The scientists point out that the lock washer model has been around for two decades and has been the guiding paradigm in the field of how a clamp is loaded around DNA. But in this case, it is wrong.

Another surprise was that the 9-1-1 clamp loader complex was observed to bind DNA in the opposite orientation to other clamp loader complexes that act on DNA undamaged during normal DNA replication. This observation explains how 9-1-1 is specifically recruited to sites of DNA damage.

From fundamental research to translational research

As well as providing a satisfying answer to a fundamental biological puzzle, Dr. Remus believes the research could eventually lead to better cancer drugs.

Many existing chemotherapy drugs work by interfering with DNA replication in cancer cells and causing the type of DNA damage that is normally repaired by 9-1-1 clamp-induced repair processes. Since cancer cells already have a reduced ability to repair DNA damage, adding DNA-damaging chemotherapy drugs can overwhelm the cells’ ability to repair their DNA, and so they die. (That’s how drugs called PARP inhibitors work, for example.)

With this new knowledge of how 9-1-1 interacts with other repair proteins and with DNA, scientists could potentially design drugs that specifically interfere with this step of the repair process, making the drugs even more effective chemotherapy.

“One of the great benefits of working here at SKI is that a scientist’s basic research can be the starting point for translational studies that ultimately lead to better treatments,” says Dr. Hite.

Reference: Castaneda JC, Schrecker M, Remus D, Hite RK. 9-1-1 checkpoint clamp loading and release mechanisms. Nat Struct Mol Biol. 2022. doi: 10.1038/s41594-022-00741-7.

This article was republished from the following documents. Note: Material may have been edited for length and content. For more information, please contact the quoted source.

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