Function of RAD51 protein in repairing double-strand DNA breaks on chromosomes clarified by the University of Tokyo — Opening a path to understanding cancer development

A research group led by Graduate Student Takuro Shioi of the Graduate School of Science at the University of Tokyo and Professor Hitoshi Kurumizaka of the Institute for Quantitative Biosciences at the University of Tokyo has announced that they have shed light on the mechanism by which the DNA repair protein "RAD51" repairs double-strand DNA breaks on chromosomes. The mechanism was clarified through analysis of the cryo-electron microscopy structures of complexes in the process of repairing double-strand DNA breaks by human RAD51 in vitro. They found that the amino-terminal domain of RAD51 binds to nucleosomes and proceeds with repair by peeling DNA wrapped in nucleosomes. As many patients with cancer have been shown to have mutations in the N-terminal domain, these results are expected to contribute to clarification of the mechanism of cancer development caused by these mutations. The results were published in the April 4 issue of the international academic journal Nature.

Genomic DNA is damaged by ultraviolet light and other factors on a daily basis, and double strand breaks due to radiation, in particular, are known to cause cancer. The DNA repair protein "RAD51" is known to be responsible for repairing DNA from such damage. Meanwhile, genomic DNA in chromosomes of eukaryotes, including humans, have a densely folded chromatin structure, and it was unclear how DNA in this state is repaired.

In eukaryotic chromosomes, genomic DNA is tightly wrapped around histone complexes to form nucleosomes, a succession of which in turn forms a chromatin structure. In this study, the research group reproduced the repair reaction of genomic DNA double-strand breaks in vitro and observed it using a cryo-electron microscope. Specifically, many images of reaction intermediates suspended in ice were acquired, and images of the same orientation were superimposed to obtain two-dimensional averaged images with increased resolution, which were further used to reconstruct the three-dimensional structure. Through this they successfully clarified the process by which RAD51 repairs double-strand breaks.

First, they observed that RAD51 binding to nucleosomes forms a ring-like structure consisting of octamers to decamers. Then, the RAD51 ring form incorporates the break site into its active center, forming a filament-like structure. RAD51 was found to bind to the cleavage sites by forming a helical structure while peeling DNA away from the nucleosome. The RAD51 ring structure was also shown to detect double-strand break sites via the L1 loop region, the active center of double-strand break repair. They also obtained images showing that RAD51 in a ring-like structure stands by on nucleosomes with no break sites.

The standby RAD51 is considered to be responsible for repair in the early stages of damage. The amino-terminal domain (N-terminal domain) of RAD51, whose function remained unknown, was found to be important for binding to nucleosomes. The N-terminal domain is absent in the bacterial RAD51 homolog (RecA), which does not have a chromatin structure but can repair double-stranded DNA. This indicates that the N-terminal domain was acquired during evolution of eukaryotes.

Amino acid mutations in the N-terminal domain of RAD51 have been identified in many patients with cancer. This study indicates the possibility that these patients develop cancer because the mutations in RAD51 interfere with nucleosome binding, preventing double-strand breaks from being repaired properly. Moving forward, they are going to clarify the mechanism by which RAD51 finds the break sites that need to be repaired.

Journal Information
Publication: Nature
Title: Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site
DOI: 10.1038/s41586-024-07196-4