Understand the amazing molecular mechanisms that reliably underlie the seemingly obvious phenomenon of cell proliferation.
How is it possible for one cell to grow into two without using 'magic' or 'elf'? What mechanisms enable the precise replication and distribution of the blueprint (DNA)? We want to uncover these mysteries using yeast cells as a model system.
All living things on Earth are made up of cells. For example, lactic acid bacteria used to make yogurt and yeast used to make bread and alcohol through fermentation are called unicellular organisms because they consist of a single cell. In contrast, organisms like us humans, which consist of many cells (in the case of humans, about 37 trillion!), are called multicellular organisms. Regardless of whether they are unicellular or multicellular, cells are the basic unit of life. Cells are classified into two types based on their internal structure. Cells in which the chromosomal DNA, the blueprint of life, is still present inside the cell are called prokaryotic cells (e.g. lactic acid bacteria), in the other hand, living organisms that are contained in an intracellular compartment separated by a membrane called the 'nucleus' are called eukaryotic cells (yeast, animals and plants).
When eukaryotic cells proliferate, they divide in an orderly fashion through a number of key events. The process of a cell dividing once is specifically called the 'cell cycle'. Incidentally, 'cancer' is a disease in which cancer cells proliferate in an unregulated manner due to an abnormality in the cell cycle control mechanism, which can be fatal to the maintenance of the organism's life system. Therefore, knowing how cell proliferation is regulated not only gives us the most basic understanding of life but also helps us to understand and overcome future diseases that may involve abnormal cell proliferation, such as cancer.
When cells divide, one cell (mother cell) becomes two (daughter cells), so the material in the cell must double before it divides. At this point, chromosomal DNA is the blueprint of life, so it is necessary for it to "double (replicate) accurately without excesses or deficiencies" and to be distributed accurately to the two daughter cells. We are particularly interested in the regulatory mechanisms of chromosomal DNA replication.
We conduct our research using yeasts, one of the model systems for eukaryotic cells. Yeast is not only an indispensable living organism in our daily lives, producing bread and alcohol through fermentation, but also a much-loved research material for many researchers. In fact, the 2001 Nobel Prize in Physiology or Medicine was awarded to three researchers in the cell cycle, two of whom were yeast researchers. Also, if memory serves, yeast was the research material for Dr Osumi's discoveries, which were also the subject of the 2016 Nobel Prize in Physiology or Medicine. The reason why yeast is so popular is that the mechanisms of cell structure and regulation of cell cycle progression are well conserved in eukaryotic cells in general, whether they are yeast or human cells. This means that findings obtained in yeast are directly applicable to eukaryotic cells as a whole. This is also the reason why we, including many researchers around the world, work with yeast.
DNA replication starts at a fixed position on the chromosome (called the replication start point). As mentioned above, it is now known that yeast cells have a wonderfully elegant mechanism for 'doubling' chromosomal DNA 'without over- or under-duplication and with precision' in a single cell division cycle. This mechanism is that the activation of the replication initiation point is,
The mechanism is based on the two-stage reaction of (1) preparation and (2) activation of the replication initiation point,
This mechanism is a combination of the fact that a protein phosphatase called 'CDK', which is the master regulator of cell cycle progression in eukaryotic cells, induces ⓐDNA replication activation and at the same time, inhibits ⓑthe preparation reaction.
The activity of CDKs fluctuates cyclically due to proper cell cycle progression. Therefore,
G1 phase: low CDK activity (ⓐⓑ: OFF), thus only (1) replication preparation response
S phase onwards: due to increased CDK activity (ⓐ: ON), (2) replication start point activation, at the same time as CDK ⓑ activity occurs, so that once activated, the preparatory reaction cannot occur again at the replication start point; CDK activity remains high until the end of M phase when cells complete division, and cells thus undergo the same This is how cells regulate DNA replication to occur only once during a single cell cycle.
In our previous work, we were the first in the world, simultaneously with a group in the UK, to elucidate how CDKs activate the replication origin (ⓐ reaction). We have also contributed to the elucidation of how CDKs inhibit preparation (ⓑ reaction).
It is known that the chromosome organization of 'cancer cells' differs significantly from that of normal cells. This indicates that defects in the mechanism for maintaining stable chromosomal DNA from one generation to the next can result in abnormal chromosome organization, causing cells to become cancerous. Moreover, as this is an organism, the control is close to 100%, but not completely 100%. Therefore, it can be thought that even normal cells escape (escape) from the mechanism that limits DNA replication to once per cell cycle at a very very low frequency. And in the long term, such phenomena may also be involved in the evolution of the organism. From this perspective, we are trying to understand the relationship between the regulatory mechanisms of DNA replication and the oncogenesis and evolution of cells.
It was mentioned above that DNA replication is initiated from replication start sites, but in fact, there are many replication start sites in eukaryotic cells (~1000 in yeast and tens of thousands in human cells). In addition, the replication initiation sites do not become active all at once during the S phase but appear to have a predetermined order of activation.
It is therefore likely that cells coordinate the activation of these many replication start sites in some way, but the details of how this works are not yet known. However, we have found that the number of factors involved in the initial activation of replication start sites is much smaller than the number of replication start sites and that the first replication start sites to which these factors bind are the first to become active. The next big question, then, is why those factors bind to those sites.
There are many other questions surrounding the regulation of DNA replication and the cell cycle, which we aim to understand one by one.