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Researchers from the Tokyo Metropolitan University and Institute of Molecular Oncology (IFOM) FIRC in Italy have successfully depleted AND-1, a key protein for DNA replication , using a recently developed system of conditional protein degradation. As a result, they have had unprecedented access to the mechanism underlying the functioning of AND-1 during DNA replication and cell proliferation in vertebrate cells, demonstrating that AND-1 performs two functions different during the replication of DNA induced by different domains of AND-1.
DNA is often referred to as the "model of life"; for living organisms to work, it is essential that all cells share the same plane. This is made possible by the process of replication of DNA, in which the DNA is copied and distributed accurately before cell multiplication. Replication is at the root of any biological heritage and relies on a whole series of biochemical pathways designed to ensure that it occurs without error and at the proper speed. Failure to do so can have catastrophic consequences, including cancer: understanding the specific mechanisms behind this extremely complex procedure is of utmost importance.
The AND-1 / Ctf4 protein is a key player in DNA replication and is found in a wide range of living organisms, from fungi to vertebrates. Ctf4 / AND-1 is essential in some organisms, but its potential for cell proliferation in vertebrates has not been experimentally demonstrated. Moreover, it is not known how the loss of AND-1 affects cell proliferation.
In order to answer this question, a team led by Dr. Dana Branzei of the IFOM and Kouji Hirota of the Tokyo Metropolitan University combined the use of two unique systems, the DT40 cell, a type of avian cell particularly suited to genetic engineering. and the auxin-inducible defron system (AID), a means of achieving selective depletion of a target protein. With these, they managed to establish the and 1-aid cell line, in which a modified version of the AND-1 protein is degraded a few hours after the addition of auxin, a type of plant hormone. This cell line allowed them to analyze the acute consequences of the loss of AND-1, thus providing an unprecedented insight into its role.
When done correctly, DNA replication should result in the formation of new double-stranded DNA helices. The authors used transmission electron microscopy (TEM) to visualize DNA replication intermediates and observed newly synthesized DNA with abnormally long single-stranded DNA at the point of forked branching. Absence of AND-1. They hypothesized that this was due to an enzyme, a nuclease, cutting DNA, disrupting the process of disassembling the strands. By subsequently adding a compound that suppresses the action of a particular nuclease, MRE11, they have succeeded in reversing the abnormal replication fork phenotype and recovering cell division, thus demonstrating explicitly the key role played by AND-1 in the prevention of nascent cleavage of DNA by the virus. nuclease during replication. Further analysis revealed that a specific part of the protein called WD40 repeats was responsible for preventing damage accumulation on the strand.
In addition to these breakthrough findings, the study highlights the successful combination of state-of-the-art techniques to achieve conditional inactivation of specific proteins; the new cells have actually been developed in a single month. This leaves the interesting perspective of the method applied to study other genes and processes that are otherwise difficult to target, which opens up new perspectives on cell function.
This work was supported by the Italian Association for Cancer Research (IG 14171 and IG 18976), the European Research Council (start-up grant 242928 and consolidation grant 682190) and a JSPS KAKENHI grant (JP16H02957). ). The study was published online in the journal Nature Communications.
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