Our health depends on the accurate transmission of genetic information. Multiple mutations, due to errors in DNA replication, DNA repair, and chromosome segregation, cause cancer. The need for multiple mutations selects for genetic instability, mutations that themselves increase mutation rates and thus contribute to the resistance of cancer to therapy. Studying the events that lead to genetic instability and the genes that mutate to cause instability is difficult in patients or animal models. We have made a diploid yeast model that allows us to select for mutations that improve cell proliferation by inactivating specific genes, thus leading to the evolution of genetic instability. The human homologs of genes that often mutate to cause instability in yeast will be candidate targets for mutations that cause genetic instability in human cancer and we will collaborate with a human cancer geneticist to follow these leads. The proposed work has three parts: 1) To examine the evolution of genetic instability in diploid yeast cells. Cells will be mutagenized and pools of mutant clones will be selected for stepwise inactivation of growth suppressor genes and activation of growth promoting genes. Experiments will find the mutations that cause genetic instability in 100 independently evolved examples of genetic instability, including selections for the activation of growth-promoting genes as well as the inactivation of growth suppressing genes. Preliminary results reveal mutations in genes that have not previously been implicated in genetic instability in yeast but have been implicated in human cancer. 2) For a selected subset of genes, chosen for their relevance to human cancer, more detailed experiments will examine mechanism of instability by characterizing the mutations that unstable strains produce and the mechanism of instability. Two initial examples will focus on the role of Holliday junction resolvases in stimulating mitotic recombination and determining whether different types of mutation that accelerate progress through G1 are equally like to cause genetic instability 3) Tumors are metabolically different from normal tissue and their cells are often starved. Preliminary experiments show that sudden glucose starvation leads to a rapid arrest of the yeast cell cycle and experiments will investigate how starvation arrests the yeast cell cycle and ask if this arrest or mutations that perturb the arrest lead to genetic instability.

Public Health Relevance

All human cancers are genetically unstable, meaning that tumor cells accumulate mutations faster than other cells in our body, and explaining why tumor cells can mutate to become resistant to cancer therapies. We have genetically engineered the baker's yeast, Saccharomyces cerevisiae, to make it a model for how genetic instability arises during the selection for mutations that allow cells to grow and divide faster. The results of our work will improve the accuracy of cancer diagnosis and identify targets for drugs that could be used to reduce genetic instability, thus improving the efficacy of existing cancer therapies.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM043987-26
Application #
9197659
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Willis, Kristine Amalee
Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Nannas, Natalie J; O'Toole, Eileen T; Winey, Mark et al. (2014) Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle. Mol Biol Cell 25:4034-48
Nannas, Natalie J; Murray, Andrew W (2014) Tethering sister centromeres to each other suggests the spindle checkpoint detects stretch within the kinetochore. PLoS Genet 10:e1004492
Hyland, Edel M; Wallace, Edward W J; Murray, Andrew W (2014) A model for the evolution of biological specificity: a cross-reacting DNA-binding protein causes plasmid incompatibility. J Bacteriol 196:3002-11
Lau, Derek T C; Murray, Andrew W (2012) Mad2 and Mad3 cooperate to arrest budding yeast in mitosis. Curr Biol 22:180-90
Nannas, Natalie J; Murray, Andrew W (2012) Complications dawn for kinetochore regulation by Aurora. Proc Natl Acad Sci U S A 109:15972-3
Murray, Andrew W (2012) Don't make me mad, Bub! Dev Cell 22:1123-5
Barnhart, Erin L; Dorer, Russell K; Murray, Andrew W et al. (2011) Reduced Mad2 expression keeps relaxed kinetochores from arresting budding yeast in mitosis. Mol Biol Cell 22:2448-57
Lang, Gregory I; Murray, Andrew W (2011) Mutation rates across budding yeast chromosome VI are correlated with replication timing. Genome Biol Evol 3:799-811
Elez, Marina; Murray, Andrew W; Bi, Li-Jun et al. (2010) Seeing mutations in living cells. Curr Biol 20:1432-7