Analysis of Scc3 postranslational modification and function in response to DNA damage

Genetic material for all cells is organised into chromosomes that must be replicated for cells division. When chromosomes replicate they consist of two genetically identical copies of the parent chromosome. Each of these copies is called a sister chromatid. For ordered segregation of chromosomes when cells divide, it is essential that sister chromatids do not separate until exactly the right moment. If they do separate early, random segregation will result causing a deleterious unequal distribution of chromosomes in daughter cells, this is aneuploidy. Holding sister chromatids together is achieved through a protein complex called made uo of proteins called cohesins. Cohesins create a link between sister chromatids that is called sister chromatid cohesion. The right time for separation of sister chromatids is after all duplicated chromosomes are aligned on the nuclear equator, with spindle fibres from opposing poles sides of the cell tugging at one sister chromatid each. At the time of nuclear division, anaphase, sister chromatid cohesion breaks down as a result of cell cycle regulated destruction of one of the cohesin proteins. This allows spindle fibres to pull sister chromatids away from each other. Studying proteins from yeast cells, we have found a common type of posttranslational modification, phosphorylation, is happening to one of the cohesin proteins called Scc3. This is a very highly conserved protein known to be phosphorylated in mammals, but in a different way. Mammalian Scc3 (or SA2) is phosphorylated as part of undoing sister chromatid cohesion, this is regulated by the cell cycle. The phosphorylation we detected in yeast Scc3 is very different because it is in response to DNA damage, and the enzymes that catalyse the phosphorylation of yeast Scc3 are well known to be part of the DNA damage response. DNA damage caused by a chemical causes Scc3 phosphorylation in mitotic cells, and more complex multiple phosphorylation events arise in the sexual cell division, meiosis. In meiosis self/inflicted DNA damage in the form of double/strand breaks stimulates genetic recombination. The extra phosphorylation we see in meiotic Scc3 is dependent on this damage. We aim to determine 1/ Which amino acids of yeast Scc3 of phosphorylated. Then, we will use molecular biology techniques to make mutant forms of Scc3. One set of mutants will be unable ot be phosphorylated in response to DNA damage. The other set of mutants will have amino acid changes that mimic the charge effects of phosphorylation, even when there is no DNA damage induced. Using these mutants we will the ask 2/ What happens to the strength of SCC in both mitosis and meiosis. 3/ If mutated cohesins are properly situated on chromosomes 4/ If mutated cohesins are properly recruited to sites of damage. 5/ Whether or not the phosphorylation are required for effective DNA repairs. Discovering how and why Scc3 influences DNA repair is very important to understanding different aspects of cell survival mechanisms. The link between chromosome stability in cells division and DNA repair is a very important one that tells us a lot about how life has evolved and how it can go wrong. It will be very interesting in the future to find out if mammalian Scc3 also responds to DNA damage.

To prevent random segregation of nascent chromatids, Sister Chromatid Cohesion (SCC) holds them together until anaphase. SCC is highly conserved in eukaryotes, the proposal uses budding yeast as a model organism to understand the interface between SCC and the DNA damage response. The SCC complex includes a little studied essential protein, Scc3. We found Scc3 is a substrate for Tel1 and Mec1 kinases, key players in the DNA damage response pathway. Initially we discovered Scc3 is phosphorylated during meiosis, probably at multiple sites and definitely in a Spo11/DSB dependent manner. Both Tel1 and Mec1 are involved in establishment and/or maintenance of phosphorylation. In vegetative cells we found Scc3 is phosphorylated in response to the MMS, though we believe fewer sites are modified than in meiosis. The preliminary data raises questions about exactly where the phosphorylation events occur on Scc3, and what is their significance. Cohesins are recruited to sites of DSBs, the exact reason for this is unknown, it could be to stabilise the chromatids or protect the undamaged sister from further assault. We will work with both vegetative and meiotic cells to determine the significance of Tel1/Mec1 dependent posttranslational modification of Scc3. Scc3 contains multiple SQ and TQ motifs that are potential substrates for Tel1 and Mec1. We will determine which are phosphorylated and whether or not our temperature sensitive allele of SCC3 is a poor substrate. Once identified, we will further mutate motifs to EQ to mimic phosphorylation and determine any significant phenotype. Phenotypes to investigate include slow growth, MMS sensitivity, failure to repair HO/endonuclease or Spo11 induced DSBs and cytological analysis of precocious sister chromatid separation. Chromatin immuno/precipitation will be used to determine the importance of Scc3 phosphorylation on cohesin recruitment to DSBs and biochemical analyses will address issues of building the SCC complex.