sicker; i.e. to test whether we can reverse the mutant phenotype of mec1-100 when the suppressor gene is ectopically over-expressed in mec1-100.

Transcription

1 Internship Report ~FMI (Basel, Switzerland)~ Kyoto University Faculty of Medicine 1 st grade Yuiko Hirata This summer I went to the FMI (Friedrich Miescher Institute for Biomedical Research) in Basel. This is a report about my experiments at the lab and life in Basel. During this stay I created mutants in Saccharomyces cervesiae (S.c) yeast cells and checked their phenotypes. I also learned making mutants by crossing mating type a and α yeast strains, for example, making a double mutant (having mutations in two different genes in a single cell) by crossing two single mutants. ---Science report--- Introduction DNA replication checkpoint has an important function in stabilizing and binding replication fork and signaling to the cell cycle machinery when replication forks encounter damaged DNA sites or are stalled. Improper functioning of this checkpoint causes the accumulation of mutations. This checkpoint pathway is highly conserved among the eukaryotes, and ATR (mammals)/mec1 (S.c) functions as a major sensor kinase to trigger this signal transduction cascade. The mec1-100 S.c mutant retains intact G2/M checkpoint but has defects in S phase checkpoint activation. In S. Gasser lab, it has been shown that the mec1-100 mutation causes a partial defect in the retention of replicative DNA polymerases at stalled forks when cells are arrested in S phase by hydroxyurea (HU), a ribonucleotide reductase inhibitor. To have a molecular insight into the role of Mec1 in the replication checkpoint and fork stability, they conducted the genetic suppressor screening with mec1-100 on the HU-induced fork arrest condition. They identified ~20 intragenic suppressors (each suppressor contains one additional point mutation in the mec1-100 gene) and 2 recessive extragenic suppressors (outside of the mec1-100 gene). Purpose The aim of my study was to test 1) if Mec1 activator, Dpb11 (TOPBP1 in mammals) or other checkpoint factors are necessary for the effect on the intragenic suppression, 2) if a gain of function of an extragenic suppressor makes mec1-100

2 sicker; i.e. to test whether we can reverse the mutant phenotype of mec1-100 when the suppressor gene is ectopically over-expressed in mec Experimental methods and Results 1. Generating dpb11-1 mutant cells in the W303 background. First I amplified the PCR fragment with which I generated dpb11-1 yeast mutant using homologous recombination mediated insertional mutagenesis in yeast cells. The dpb11-1 mutation is a C-terminal truncation of Dpb11 (generating stop codon resulting in a deletion of amino acid ), and confers a temperature sensitive phenotype at 37 C and sensitivity to the drug that causes the replication fork stalling, such as HU and MMS (methyl methanesulfonate). Following the protocol, transformation of yeast cells, I transformed wild type cells by inserting the PCR product I made previously. After making a master plate from 5 colonies, I checked them by colony PCR, and found that 3 out of 5 turned out to be the ones we aimed for. To confirm the dpb11-1 mutation of the three yeast clones, we also checked their phenotype by the spot assay (to see HU sensitivity and temperature sensitivity), and also conducted the sequencing of the PCR amplified endogenous Dpb11 gene from the three yeast clones. For this sequencing, I first tried to make fragments by colony PCR, but it did not work (It was not clear why this did not work.). Then I prepared the genomic DNA of the three yeast clones, and used it as a template. I successfully amplified the dpb11-1 fragments from the three clones and purified the PCR products by fractionating in the agarose gel electrophoresis. After measuring the concentrations of the DNA fragments and adjusting them to a concentration suitable for base sequencing, I was able to submit them to be sequenced by a technician in a core facility of FMI. The DNA fragments derived from two out of the three yeast clones were successfully sequenced. I confirmed that both of them carried the aimed mutation. Thus, I was able to stock these two independent dpb11-1 mutants. 2. Creating double mutant cells In the first series of the experiment, I wanted to combine an intragenic mec1-100 suppressor mutation with one of the following five mutations (rad9δ, chk1δ, mrc1δ, rad24δ, and tel1δ) in individual yeast cells. The method of creating such doublemutant cells is common to every combination of two mutations, which first starts from streaking out cells, mating them on a plate, and then streaking out the mated

3 cells on each suitable selection plate. After I followed this experimental method, I picked up a few colonies, streaked them out on culture plates, and then replicated them on sporulation plates. On sporulation plates, or, upon starvation, yeast cells undergo the process of meiosis and create spores. I dissected resulting individual spores, checked the marker gene that associates the individual mutantions (genotyping), and thereby identified the spore that carried two expected mutations, such as the intragenic mec1-100 suppressor mutation together with the rad9δ mutation. In this way, I was able to create 10 double mutants (5 double mutants with either mating type a or α). In the second series of this multiple-gene-disruption experiment, I crossed rad24δ cell with 6 different intragenic mec1-100 suppressors. I was able to generate all the double mutants I had aimed to make in this experiment. In the third series, I crossed dpb11-1 (which I had made previously) with the same 6 different mec1-100 intragenic suppressors as above, and with a pure mec1-100 (without suppressor mutation). I was able to generate the double mutants that carry both mec1-100 intragenic suppressor and dpb11-1 mutations. However I was unable to obtain any mec1-100/dpb11-1 double mutants (no double mutant out of 51 total viable spores from the cross). From this observation, we concluded that mec1-100 and dpb11-1 are synthetic lethal, and that the intragenic suppressors restores the viability of mec100/dpb11-1. I also checked the sensitivity of the double mutants, which I obtained above by the spot assay on HU plates. I demonstrated that tel1 or chk1 deletion has no impact on the mec1-100 suppression, whereas rad24 or dpb11-1 reduces the viability of mec1-100 suppressors I tested. Interestingly, among the different suppressors, we saw different degrees of Dpb11 dependency for the HU viability, indicating that there are different means or levels of mec1-100 suppression. In the forth series, I tried to introduce the POL2 (a component of DNA polymerase ε)-13myc tag gene into 7 different mec1-100 suppressor mutants by crossing. I was not able to finish generating all strains before the end of my internship, and the supervisor, Dr. K. Shimada took over this project and successfully generated all mutants. 3. Gateway cloning of extragenic suppressors and expression of them in mec1-100 cells (due to intellectual property, I am not able to describe the gene name of two

4 extragenic suppressors in this report. Hereafter I refer to the two suppressor genes as X and Y.) This may have been the most interesting experiment of all for me. First I used the Gateway cloning kit to create an entry plasmid clone carrying gene X or Y. I tried to clone those genes into the donor vector included in the kit by using the BP reaction from the Gateway clone we already had. However, I got only a few colonies. Moreover, I did colony PCR and thereby figured out that no clones included an expected insert fragment. At first we suspected that the entry plasmid had some problem. But I did not find any degradation or low quality of the plasmid that I used. Dr. Shimada and I could not figure out what the cause of this failure was, but there may have been certainly something wrong with one of the BP reagents included in the Gateway cloning kit. I tried the same BP reaction over again with a different donor vector, and fortunately, the experiments finally worked out this time, indicating that the firstly-used donor vector had not worked. I was then able to generate the X and Y genes integrated in the entry plasmid and stocked the resulting plasmids. Next I used the LR reaction to subclone X and Y from the entry clones into 2 destination vectors. I successfully produced 4 expression clones. Using these expression plasmids, I transformed and obtained wild-type and mec1-100 cells, which carry galactose inducible X and Y genes on each plasmid. To see the phenotype of the transformed yeast cells, I did a spot assay on non-inducible (2% glucose) condition with or without 20 mm HU and inducible condition (2% galactose) with or without 5, 10, or 20mM HU. Unfortunately I did not have enough time to finish this experiment, but Dr. K. Shimada took over and sent me the results. I would like to hereafter describe the outcome of this experiment. The extra-copy of both X and Y increased the HU sensitivity of mec1-100 compared to the control vectors, even in the non-inducible condition. This suggests that even a very low excess amount of X and Y molecules sensitizes the mec We also found that overexpression of X and Y itself is very toxic to the cells (even for the wild-type cells). Next, Dr. Shimada tested effects of various levels of X and Y expression on cells by changing the carbon source ratio., and found that the ectopic expression of X and Y elevates the HU sensitivity of mec We were therefore able to demonstrate that excess amounts of X and Y

5 could reverse the situation where mec1-100 cells become viable on HU in the loss of X or Y gene function. This is a way to confirm the phenomenon that recessive X or Y mutant suppresses mec Others I tried several times to get a PCR fragment that carries ACT1 mutant in a plasmid, but I did not succeed. I also tested newly generated two anti-mec1 antibodies to see if they worked. I loaded the total cellular proteins from wild type, mec1δ, mec1-100, and Mec1-19Myc tag onto SDS gel electrophoresis, and carried out western blot analysis. However, I failed to detect specific Mec1 signals using these antibodies. Combined with other people s results, it is not very likely that we are able to detect Mec1 by these new antibodies. Acknowledgements To the whole Gasser lab in FMI, and especially to Dr. Kenji Shimada, who supervised me and taught me everything. He has also contributed to my work on writing this report by giving me much information. Thank you very much. ---Life in Basel--- The FMI was a wonderful institute. Although I did not have many opportunities to make use of them, they had the best microscopes and the latest equipment. Also anyone would realize the cleanliness of the labs and building. Most of all, there were brilliant people, who were all very cooperative. They would be happy to help you out whenever you had a problem or you needed something. I thought that it was very nice and efficient that you could go by another lab, say hi, and get the information you need from the professionals of that field of research. The support and treatment for visiting people, even for a person like me, was plenty and generous. The food at the cafeteria in the campus was great, and the environment of the campus and location was good. The bread and breakfast home we stayed at was a very comfortable place, with a beautiful garden around it. We could enjoy the nice Swiss atmosphere there. Above the fact that it was a beautiful quiet place, there was absolutely no problem living there. There was hot water, a nice kitchen, cleaning, and we could use the washing machine. It was right near the bus stop so it was convenient enough to go

6 around by public transportation. I would recommend everyone to buy the half fare ticket or Swiss pass especially if you have plans to travel around Switzerland, because transportation fee is much more than you expect! The owner of the house and people in the house were generous, and cared for us very much. Basel is a large city in the German-speaking region of Switzerland, and it is near the point where Switzerland, Germany and France share their borders. Accordingly, you can visit not only Switzerland but also many other places in Germany and France on the weekends. I myself went around 4 countries, including Monaco. It is very nice to be able to see different cultures, visit different museums and do many other things. I realized that culture depends much on the local language, by traveling around Switzerland. It was very interesting to see some part of France, some part of Germany (but with Swiss taste) in the same country. Life in Basel was mostly easy and comfortable, but the high price may be a heavy burden. But I told myself that it might be the one and only chance, and did not hesitate to travel around, eat local food, and so on. I regret that I hadn t brought a swimming suit, which would have been very useful, for swimming and rafting in the Rhein must have been wonderful. The month and a half in Basel was a marvelous and fruitful time, and I am sure it has somewhat influenced my future prospects. I would like to thank everyone that supported me, and Professor Takeda who gave me such a wonderful opportunity.