* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download overexpression of mcm protein potentially causes cancer
Genome (book) wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
SNP genotyping wikipedia , lookup
DNA polymerase wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Genetic engineering wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Primary transcript wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Epigenomics wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Microevolution wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Non-coding DNA wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Genomic library wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
DNA supercoil wikipedia , lookup
Molecular cloning wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Point mutation wikipedia , lookup
DNA vaccination wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Helitron (biology) wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Oncogenomics wikipedia , lookup
History of genetic engineering wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
OVEREXPRESSION OF MCM PROTEIN POTENTIALLY CAUSES CANCER Yujie Mu, Anthony Schwacha Department of Bioengineering and Department of Biological Science, the University of Pittsburgh INTRODUCTION In the United States 2015, the total population of new cancer cases reaches to 1.6 million, within which around 0.6 million deaths are projected to occur. [1] From data estimated by International Agency for Research on Cancer, there are 14.1 million new cancer cases in 2012, and 8.1 million cancer deaths occurred worldwide. [2] In recent years, cancer becomes one of the most fatal diseases threatening human health. Targeted therapy, one of the most common treatments for the cancer, targets the small molecules in cancer cells that help them grow, divide and spread. [3] Hence, small molecular inhibitor becomes a new promising treatment to cancers under targeted therapy category. In order to come out with a more effective and novel molecular inhibitor to treat cancer, it is important to first understand mechanisms of cancer development and how it is affected by small molecular inhibitors. Cancer results from a corruption of an organism’s genetic code (genomic instability), and mistakes made during DNA replication are a particularly prevalent cause of such instability. The cause of genomic instability can be attributed to defects in genes required for nucleotide repair, double-strand DNA break recognition and repair, genetic recombination and mismatch repair, which also associate with human cancer syndrome. [4] The previous study established the possibility that alterations to one particular replication factor – the Mcm2-7 helicase – might contribute to genomic instability. Mcm2-7 is formed from six different subunits (labeled 2 through 7) and serves to unwind duplex DNA in advance of DNA polymerase during DNA replication progress. [5] My lab and others have shown that Mcm2-7 is also involved in various regulatory processes that ensure accurate DNA replication. [6] Interestingly, human tumors commonly contain mutations in at least one of the Mcm subunits, and many overexpress individual subunits. OBJECTIVE The goal of my research is to test if Mcm subunit overexpression can lead to genomic instability that potentially triggers cancer, or if in contrast the observed Mcm mutations are simply a result of cancer development. HYPOTHESIS/SUCCESS CRETERIA I hypothesize that there is at least one Mcm subunit overexpressed causes significant genomic instability since overexpression of Mcm protein will break up the balance in reacting with other regulatory molecules. We designed three success criteria aiming to achieve more confidential results and set the standard cutoff analyzing data. Firstly, we need to construct target Mcm subunit gene under galactose promoter and insert appropriate selectable marker to strains. Secondly, we set mcm2DENQ already known to cause genomic instability as experimental positive control. The results of 2DENQ are considered as statistical significant number, which determines whether overexpression of Mcm subunit causes genomic instability. Thirdly, we will perform each experiment for three times to increase reliability and minimize statistical errors. METHOD Fundamental replication processes are completely conserved between yeast and humans; therefore yeast provides a good human disease model, especially for cancer study. As Mcm2-7 complex is highly conserved among all eukaryotes, we will be conducting experiments using budding yeast as a highly tractable model system. [7] Mcm subunits have around 50% protein sequence identity between human and budding yeast by testing genomic stability following conditional overexpression of individual Mcm subunits. [5] Genomic instability can be determined by whether it causes both DNA-level instability and chromosome-level instability by examining the fraction of single-strand and double-strand DNA breaks and the rate of plasmid loss per generation respectively. To overexpress individual Mcm subunits, an appropriate genetic construction will be stably transformed into 6 strains that will place each individual Mcm gene under the high-level inducible expression of the galactose promoter; thus cells grown on glucose will contain normal levels of Mcm proteins, while cells grown on galactose will overexpress the target Mcm subunit. As a cytological assay for DNA instability, we will use a reporter strain that contains an in-frame fusion between green fluorescence protein (GFP) and RPA, which is a protein specifically required to repair DNA single strand breaks. In this assay, cells lacking DNA damage will contain a diffuse fluorescence throughout the entire nucleus corresponding to RPA-GFP, where as cells with DNA breaks will localize this protein to break sites and form sharply defined point (foci). Similar to procedures of RPA-GFP assay, Rad52-GFP assay is designed to localize double-strand DNA breaks sites. The test strains will be grown in the appropriate media, and the percent of cells containing RPA-GFP or Rad52-GFP foci will be scored using fluorescence microscopy. Plasmid loss is described as an event where daughter cell does not obtain one copy of plasmid from mother cell during replication. [8] Normally, yeast will replicate and segregate plasmids with good efficiency. Genetic alteration that reduces the ability of cells to perform either DNA replication or chromosome segregation will generate cells at a higher rate of plasmid loss. We perform this assay by constructing ADE3, which is a color indicator, into the circular DNA. Cells with plasmid and ADE3 gene translated show red color. Otherwise, cells shown white are considered to lose plasmid. Then we follow the previous established protocol to calculate the rate of plasmid loss by each generation. Reaching to cutoff, overexpression of specific Mcm subunit is counted to cause chromosome instability. RESULTS RPA-GFP assay and Rad52-GFP assay are performed with three trials, and calculated values for plot comparison are reported as the average across all subjects. The purple bar indicates percent of foci induced by mcm2DENQ positive control as significant cutoff value. The wildtype strains grown in glucose are shown in blue bars, and overexpressed strains grown in galactose are shown in red bars. Figure 1. Mcm subunit overexpression caused single-strand DNA damage. The indicated strains were scored for RPA-GFP foci following growth in either glucose or galactose containing media. The numbers shown reflect the percent of cells in the population containing 1 or more RPA-GFP foci with standard deviation ±2~3%. Figure 2. Mcm subunit overexpression caused double-strand DNA damage. The indicated strains were scored for Rad52-GFP foci following growth in either glucose or galactose containing media. The numbers shown reflect the percent of cells in the population containing 1 or more Rad52-GFP foci with standard deviation ±1~2%. Plasmid loss assay was performed with two trials, and all values are averaged in the calculation. There is no control group for this assay. Rate of plasmid loss can be scored under 5% generally, with which significant cutoff can be considered as three times of plasmid loss rate of wildtype. Figure 3. Mcm subunit overexpression caused chromosome instability. The indicated strains were scored for plasmid loss rate of each generation following growth in either glucose or galactose containing media. The numbers shown reflect the percent of plasmid loss rate with respect to wildtype plasmid loss rate with standard deviation ±3~4%. The results of experiments support my hypothesis. Overexpression of Mcm3, 6 and 7 causes significant singlestrand DNA breaks (~55%, 26% and 51% respectively), compared with RPA-GFP foci of 2DENQ (~25%). Overexpression of Mcm3 and 7 causes significant double-strand DNA breaks (~44% and 22% respectively), as 2DENQ shows around 18% defect. Overexpression of Mcm5 (~17%) and Mcm7 (~14%) has comparatively significant plasmid loss rate than that of wildtype (<5%). DISCUSSION My success criteria were meet, except for repetition of plasmid loss assay. The results for single-strand and doublestrand DNA breaks are consistent between Mcm3 and Mcm7 that these two subunits significantly cause DNA instability, while Mcm6 is somehow consistent but smaller effect on double-strand DNA breaks. The limitation of my research is that counting foci and cells under microscope is subjective that varies from person having different counting rules, and there is no existing advanced method to count cells in a more effective and accurate way. In conclusion, the subunit Mcm7 is consistent in above three assays that it causes both DNA instability and chromosome instability. Mcm7 causes genomic instability that potentially triggers cancer. For future study, experiments are in progress to test if over expression of either Mcm3 or Mcm7 causes other types of DNA damage, as well as to test various hypotheses for why overexpression of either of these subunits would result in DNA damage. ACKNOLEGMENT I would like to thank Dr. Anthony Schwacha for mentoring my research, and Jennifer Liberato, Elle Fernander, and Julia Verbier for construction of some of my strains. REFERENCES 1. Siegel, RL1, and KD Miller. "Cancer Statistics, 2015." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 2 Nov. 2015. 2. Torre, L. A., Bray, F., Siegel, R. L., Ferlay, J., LortetTieulent, J. and Jemal, A. (2015), Global cancer statistics, 2012. CA: A Cancer Journal for Clinicians, 65: 87–108. doi: 10.3322/caac.21262 3. Chemotherapy. (2015, April 29). Retrieved December 6, 2015, from:http://www.cancer.gov/aboutcancer/treatment/types/chemotherapy 4. Preston, B., Albertson, T., & Herr, A. (n.d.). DNA replication fidelity and cancer. Seminars in Cancer Biology, 281-293. 5. Bochman, L.A., Schwacha, A. (2009). “The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase.” Microbiology and Molecular Biology Reviews, Dec. 2009. p.652-683. 6. Forsburg, S. (2004). Eukaryotic MCM Proteins: Beyond Replication Initiation. Microbiology and Molecular Biology Reviews, 109-131. 7. Botstein, D. (1991). Why yeast? Hospital Practice. 8. Miller, C. “ Loss of Antibiotic Resistance in Escherichia Bacteria.”