IMAGE: CtIP / Ctp1 stimulates the endonuclease activity of the MRN complex, which not only polishes “dirty” ends of DNA breaks, but also promotes the precise repair of DNA breaks through the homologous recombination mechanism. view More
Image credit: PNAS
Scientists at the Tokyo Institute of Technology (Tokyo Tech) have discovered mechanisms that underlie the activation of the MRN complex – the cell’s DNA scissors. Using purified yeast proteins, they showed that the phosphorylation of Ctp1, a homologue of a tumor suppressor protein, plays a key role in activating the DNA clipping activity of the MRN complex. Interestingly, a short segment of yeast Ctp1 or its human counterpart could stimulate the endonuclease activity of their respective MRN complexes, indicating its conserved function across species.
DNA acts as a roadmap that controls the identity and functions of cells. A defect in DNA can have serious deleterious effects, resulting in malfunction or loss of important proteins, thereby affecting normal cell function and viability. These disorders often manifest as double-stranded breaks in DNA, which can occur spontaneously or as a result of the action of certain chemicals. To deal with these kinks, cells have developed DNA repair machinery that scans, identifies, and fixes breaks in DNA by ligating the gaps. However, DNA breaks often have “dirty ends” that cannot be ligated or sealed directly because they are not exposed or blocked by certain proteins or irregular chemical structures. Such DNA ends must therefore first be cut off and released so that they can be processed further. In addition, such a final resection of broken DNA ends is a prerequisite for them to be precisely repaired by homologous recombination. Among such molecular scissors or nuclease enzymes, Mre11 plays a key role.
Mre11 together with the proteins Rad50 and Nbs1 together form the MRN complex. It has been shown that the interaction of this complex with the tumor suppressor protein CtIP triggers the DNA clipping function of the complex in humans (Figure 1). However, the mechanisms underlying this interaction have so far remained unexplored.
Now, Assistant Professor Hideo Tsubouchi and Professor Hiroshi Iwasaki from the Tokyo Institute of Technology and their team have decoded the step-by-step interaction and activation of the MRN complex using Ctp1 proteins in yeast that are homologous to human CtIP. Iwasaki discusses her results, which were recently published in PNAS: “The MRN complex is critical for the homologous recombination-mediated repair of DNA double-strand breaks. To better understand how CtIP affects the activity of the MRN complex, we purified yeast proteins and quantified their interactions. “
The scientists found that phosphorylation, or the addition of phosphate groups to Ctp1, was the first important step in activating the MRN complex. In particular, the phosphorylation enabled the physical interaction of Ctp1 with the Nbs1 protein of the complex, which was of crucial importance for the subsequent endonuclease stimulation. The DNA clipping activity was extremely poor when the MRN complex was mixed with non-phosphorylated Ctp1.
In addition, the scientists identified a short stretch of only 15 amino acids at the C-terminal region of Ctp1, which was essential for the endonuclease activity of the Ctp1-stimulated MRN. In addition, a synthetic peptide mimicking this region of Ctp1 or CtIP could activate the yeast or human MRN complex, suggesting that the function of the C-terminal Ctp1 is likely to be preserved across species and is the ultimate determinant of MRN -Activation is.
Excited about the prospective application of their results, Tsubouchi notes, “The modification of the CT15 peptide can result in a potent activator or potential inhibitor of the MRN complex. Targeting this endonuclease activity can have potentially useful applications in homologous recombination-based gene editing.”
With the rapid advances in recombinant DNA and molecular medicine, these findings could enable geneticists to unlock the secrets of the genome and identify the hidden intricacies of genetic disorders more easily and effectively in the days ahead.
Help for a helper: uncovering how different proteins work together in DNA repair https://www.titech.ac.jp/english/news/2020/046793.html
Scientists unravel the molecular details of DNA recombination regulation https://www.titech.ac.jp/english/news/2018/042590.html
Iwasaki Laboratory http://www.iwasakilab.bio.titech.ac.jp/cgi-bin/wp/english-version-of-the-front-page/
About the Tokyo Institute of Technology
Tokyo Tech is at the forefront of research and higher education as the leading science and technology university in Japan. Tokyo Tech researchers excel in areas that range from materials science to biology, computer science and physics. Tokyo Tech was founded in 1881 and is home to over 10,000 undergraduate and graduate students annually who develop into scientific leaders and some of the most sought-after engineers in the industry. The Tokyo Tech Community embodies the Japanese philosophy of “Monotsukuri”, which means “technical ingenuity and innovation,” and strives to contribute to society through effective research. https://www.titech.ac.jp/english/
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