Researchers at the Tokyo Metropolitan University have studied DNA repair through homologous recombination, where the RecA protein repairs breaks in double-stranded DNA by incorporating a dangling single-strand end into intact double strands and repairing the break based on the undamaged sequence. They found that RecA determines where the single strand should be placed in the double helix without uncoiling it by even a single turn. Their findings promise new avenues in cancer research.
Homologous recombination (HR) is a ubiquitous biochemical process common to all living organisms, including animals, plants, fungi, and bacteria. In everyday life, our DNA is exposed to all kinds of environmental and internal stresses, some of which can lead to breaks in both strands of the double helix. This can be catastrophic and can lead to imminent cell death. Fortunately, processes like HR continuously repair this damage.
During HR, one of the two exposed ends of the break in the helix falls off, exposing a single-stranded end; This is called resection. Then, a protein called RecA (or an equivalent protein) binds to the exposed single strand and an intact double strand nearby. Next, the protein „searches“ for the same sequence. Once it has found the right spot, it combines the single strand back into the double helix in a process called strand invasion. The broken DNA strand is then repaired using the existing DNA as a template. HR enables the precise repair of double-strand breaks as well as the exchange of genetic information, making it an important part of biodiversity. However, the exact biochemical picture of HR, including what happens when RecA carries both single and double strands, is still unclear.
A team led by Professor Kouji Hirota of the Tokyo Metropolitan University has studied DNA repair mechanisms such as HR. In their recent work, they tried to test two competing models for what happens when HR occurs. In one case, RecA unwinds a section of the double strand during „homology search,“ trying to find the right spot for strand invasion. In the second case, there is no unwinding after RecA binds; unwinding only occurs when strand invasion takes place.
The team collaborated with a team from the Tokyo Metropolitan Institute of Medical Science to find out which of these actually happens. In the first case, they used a mutant RecA that cannot separate the double strands, to see if this affects DNA repair. It turns out that this has only minimal impact. In the second step, they tried to measure how much torsion was generated in the different phases of the process in the strand. They found that the only torsion they could detect due to unwinding occurred after homology search was completed, that is, when strand invasion occurred. The team clearly demonstrated that the second model was correct.
Detailed insights into homologous recombination are crucial to understanding what happens when something goes wrong. For example, factors involved in breast cancer (BRCA1 and BRCA2) are also responsible for correctly loading single-stranded DNA on RAD51, the human version of RecA. This suggests that issues with HR could be the cause of a high incidence of breast cancer in patients with hereditary defects in BRCA1 or BRCA2. The team hopes that insights like theirs will open new avenues in cancer research.
This work was supported by JSPS KAKENHI Grant Number JP22K06335.
Tokyo Metropolitan University
Shibata, T., et al. (2024). Homology recognition without double-stranded DNA strand separation during D-loop formation by RecA. Nucleic Acids Research. doi.org/10.1093/nar/gkad1260.