Cell cycle control of homologous recombination by the Sae2 protein in Saccharomyces cerevisiae

Each living organism is defined by a set of information that is inherited from its ancestors in the form of genes that are encoded by a specific kind of molecule called DNA. Therefore, the DNA comprises the handbook for all the things the organism may need to succeed in its environment. Because of its fundamental importance for survival, cells have developed a series of mechanisms to ensure both the stability of the DNA molecule and the proper transfer of the information to the next generation. In order to ensure that daughter cells get all the information they need, each cell undergoes a complex growth and division cycle: the cell cycle. Such a cycle starts with a cell that has just a single copy of the genetic information (DNA) that, once some parameters are fulfilled, becomes exactly duplicated. Then, and only if the DNA is completely duplicated and is undamaged, the cell divides to form two newborn daughter cells, each of them with a single copy of the genetic information. These cells can then re-start the cell cycle and continue growing and dividing. One important fact is that the cell will never progress through the cell cycle if its DNA is somehow damaged. There are many causes of DNA damage, which these include radiation, sunlight and environmental chemicals; and even the oxygen we breathe can result in damage to the DNA in our cells. Once the DNA is damaged, the cell will try to repair it. As there are many different kinds of damage, there are many different kinds of DNA repair mechanisms and, in some cases, more than one repair pathway can deal with a specific type of DNA damage. It is important to notice that not all the repair pathways are equally accurate and some of them may even leave errors in the DNA code. Therefore, the regulation of which mechanism is better to repair a lesion at each given moment is extremely important. A good example of the above types of regulation has become clear from research into the repair of the most dangerous form of DNA damage; the DNA double-strand break. There are two methods to repair such damage. The first one (end-joining) is a low accuracy mechanism that consists in direct rejoining of both ends of the molecule. The second one, a much more accurate and complex one called homologous recombination, only occurs when the cell has already duplicated its DNA and, therefore, the second copy of the DNA can be used as a donor of information. Activation of this pathway when the DNA has not been yet duplicated is, in fact, highly deleterious to the cell and can lead to its death. This means that there needs to be crosstalk between the cell cycle and the DNA repair pathways to ensure that homologous recombination is only activated once the cells have duplicated their DNA. Our research is focused on understanding the nature of this crosstalk. Using the baker's yeast Saccharomyces cerevisiae as a model organism, we have found that depends, at least partially, on the regulation of a protein called Sae2, that is activated only once the DNA has been copied. Activation of Sae2 is essential for DNA repair by homologous recombination. In order to further understand how Sae2 is acting, we present this proposal with the following major objectives: 1. We will determine how Sae2 regulates the balance between the two repair pathways (end-joining and homologous recombination). 2. We will identify other factors that help Sae2 in its regulatory roles. 3. We will identify other ways that homologous recombination is controlled during the cell cycle. 4. We will extend our work to study meiosis: the specialized type of cell cycle that produces the cells that are essential for sexual reproduction. In addition, research carried out by another member of our laboratory has identified a human protein that appears to function in the same way as yeast Sae2. Therefore, we will collaborate with him to study similar control mechanisms in human cells.

Double-strand breaks are repaired by two alternative mechanism: non-homologous end-joining (NHEJ) and homologous recombination (HR). The latter is a highly accurate mechanism that use a homologous sequence, the sister chromatid, as a donor of information, and it results in an accurate repair. However, when the sister chromatid is absent, in G1 or G0, repair by HR can lead to chromosomal rearrangements because spurious repetitive sequences can be used as donor template. For this reason, HR is actively regulated during the cell cycle and only occurs when the DNA has been replicated, in either S or G2. Although it is known that HR is controlled by phosphorylation events by cyclin-dependent kinases (CDKs) the relevant protein substrates have not yet been identified. Through our recent work, we have discovered that CDK activity controls DNA end resection, the first step of HR, by activating the protein Sae2. In our proposed work we will use a set of mutations in the SAE2 gene that affect CDK-mediated activation of the protein. The major aims and the proposed approaches to be taken to are: 1 We will determine how Sae2 regulates the balance between NHEJ and HR by combining sae2 mutations with end-joining mutants and then analyzing the phenotypes of the resulting strains and the types of repair events taking place within them. 2 We aim to identify other members of the cell cycle-dependent regulatory pathway that act together with Sae2 to control DNA end resection and HR by screening for and characterizing suppressors of sae2 point mutants. 3 We aim to identify other points of cell-cycle control of HR by in silico identification of putative CDK substrates involved in HR and characterization of non-phosphorylatable mutants of these. 4 We will determine the role of CDK-dependent activation of Sae2 in meiotic recombination by checking meiotic progression in sae2 mutants. This will be done at the genetic level and also by studying meiotic recombination by Southern blot.