Molecular Biology Faculty
Alison E. Gammie
Lewis Thomas Lab, 334
DNA Mismatch Repair & Cancer
Lynch Syndrome, also known as hereditary non-polyposis colorectal cancer, is a leading cause of inherited cancer mortality in the United States. Approximately 2-7% of colorectal cancer cases are the consequence of Lynch Syndrome, a dominant and highly penetrant disease afflicting individuals at an early age. While colorectal is the most common form of cancer found in Lynch Syndrome families, endometrial, ovarian, stomach, small intestine, liver, gallbladder ducts, upper urinary tract, brain, skin, and prostate cancers are all associated with the syndrome.
Lynch Syndrome cancers as well as many sporadic tumors a consequence of defects in mismatch repair; for example, ~17% of all colorectal cancers originate from defects in mismatch repair. DNA mismatch repair is a conserved mechanism that significantly contributes to the accurate preservation of genetic material. Mismatch recognition is accomplished in eukaryotes by MutS heterodimers in which Msh2 is the invariant partner. Mismatch binding initiates subsequent events including cleavage and excision of the error-containing strand followed by new synthesis and ligation. Without mismatch repair, nuclear DNA acquires numerous mutations, including single base substitutions and insertions/deletions at microsatellites. The increased rate of mutation often results in tumor formation in animal cells. For example, a Lynch Syndrome individual has a 80-90% chance of getting cancer before the age of 50. Of clinical significance, mismatch repair defective tumors are associated with increased resistance to certain conventional chemotherapies.
Germline mutations in either hMSH2 or hMLH1 underlie the majority of Lynch Syndrome cases. Approximately 20% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 935 amino acids. Our laboratory characterizes clinically-identified MSH2 missense mutations, using yeast as a model system. To date, the missense mutations leading to loss of mismatch repair defined important structure-function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. One area of our current research focuses on the regulatory factors contributing to a loss of mismatch repair with an aim to identifying molecules that restore mismatch repair in defective cells. In the current era of personalized medicine, our research aims to uncover the molecular defects of clinically significant variants of mismatch repair proteins to propose unique approaches to treating tumors.
Mismatch Repair & Strand Specificity
DNA mismatch repair is a highly conserved process contributing to the fidelity of genetic material. Mismatch repair includes identification of a mismatch in the DNA helix, followed by cleavage and excision of the error-containing strand. After the error is removed, a new DNA strand with correct base pairing is synthesized. One area of active research is directed toward answering two central questions about the mechanism of mismatch repair in eukaryotes: (A) How does the mismatch repair system accomplish strand specificity of repair during replication? (B) How do mismatch recognition proteins efficiently survey the nucleosome packaged genome? Our research draws on genomic, molecular, genetic, and biochemical techniques using Saccharomyces cerevisiae - an ideal organism for studying eukaryotic mismatch repair because of the ease of manipulation and its significant homology to the human system. We combine chromatin immunoprecipitation and custom tiling microarrays or high throughput sequencing to address these questions in DNA mismatch repair that were previously impossible to study in vivo.
Mutation Accumulation In DNA Mismatch Repair Defective Cells
Mutations in DNA are the fundamental drivers of evolution, genome organization and many diseases. We use next generation, high throughput DNA sequencing to address questions surrounding mutation rates, mutation accumulation context, and mutation spectra that have been difficult to answer with older technologies. Specifically, (1) what is the mutation rate in normal cells and cells lacking DNA mismatch repair, (2) do mutations occur randomly across the genome or are there chromosomal contexts that contribute to increased mutation rates? (3) what types of mutations accumulate and does the absence of DNA mismatch repair change the mutation spectra.
Lang GI, Parsons L, Gammie AE. (2013) Mutation rates, spectra, and genome-wide distribution of spontaneous mutations in mismatch repair deficient yeast. G3. [Epub ahead of print]
Arlow T, Scott K, Wagenseller A, Gammie A. (2013) Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2. Proc Natl Acad Sci. 110: 246-51. Pubmed
Tennen RI, Haye JE, Wijayatilake HD, Arlow T, Ponzio D, Gammie AE. (2013) Cell-cycle and DNA damage regulation of the DNA mismatch repair protein Msh2 occurs at the transcriptional and post-transcriptional level. DNA Repair 12: 97–109. Pubmed
Naka H, Chen Q, Mitoma Y, Nakamura Y, McIntosh-Tolle D, Gammie AE, Tolmasky ME, Crosa JH. (2012). Two replication regions in the pJM1 virulence plasmid of the marine pathogen Vibrio anguillarum. Plasmid 67: 95-101. Pubmed
Hayes AP, Sevi LA, Feldt MC, Rose MD, Gammie AE. (2009) Reciprocal regulation of nuclear import of the yeast MutSalpha DNA mismatch repair proteins Msh2 and Msh6. DNA Repair (Amst). 8: 739-751. PubMed
Gammie AE. (2008) Ultrastructural analysis of cell fusion in yeast. Methods Mol Biol. 475: 197-211. PubMed
Gammie AE, Erdeniz N, Beaver J, Devlin B, Nanji A, Rose MD. (2007) Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae. Genetics 177: 707-721. PubMed
Lahav R, Gammie AE, Tavazoie S, Rose MD. (2006) The transcription factor Kar4p has a global role in regulating the yeast pheromone response pathway. Mol Cell Biol 27: 818-829. PubMed
Gammie AE, Erdeniz N. (2004) Characterization of pathogenic human MSH2 missense mutations using yeast as a model system: a laboratory course in molecular biology. Cell Biol Ed 3: 31-48.
Fitch PG, Gammie AE, Lee DJ, de Candal VB, Rose MD. (2004) Lrg1p Is a Rho1 GTPase-activating protein required for efficient cell fusion in yeast. Genetics 168: 733-746. PubMed
Gammie AE, Rose MD. (2002) Assays of cell and nuclear fusion. Methods Enzymol 351: 477-498. PubMed
Gammie AE, Stewart BG, Scott CF, Rose MD. (1999) The two forms of karyogamy transcription factor Kar4p are regulated by differential initiation of transcription, translation, and protein turnover. Mol Cell Biol 19: 817-825. PubMed
Miller RK, Heller KK, Frisen L, Wallack DL, Loayza D, Gammie AE, Rose MD. (1998) The kinesin-related proteins, Kip2p and Kip3p, function differently in nuclear migration in yeast. Mol Biol Cell 9: 2051-2068. PubMed
Gammie AE, Brizzio V, Rose MD. (1998) Distinct morphological phenotypes of cell fusion mutants. Mol Biol Cell 9: 1395-1410. PubMed
Brizzio V, Gammie AE, Rose MD. (1998) Rvs161p interacts with Fus2p to promote cell fusion in Saccharomyces cerevisiae. J Cell Biol 141: 567-584. PubMed
Kurihara LJ, Stewart BG, Gammie AE, Rose MD. (1996) Kar4p, a karyogamy-specific component of the yeast pheromone response pathway. Mol Cell Biol 16: 3990-4002. PubMed
Brizzio V, Gammie AE, Nijbroek G, Michaelis S, Rose MD. (1996) Cell fusion during yeast mating requires high levels of a-factor mating pheromone. J Cell Biol 135: 1727-1739. PubMed
Gammie AE, Rose MD. (1995) Identification and characterization of CEN12 in the budding yeast Saccharomyces cerevisiae. Curr Genet 28: 512-516. PubMed
Gammie AE, Kurihara LJ, Vallee RB, Rose MD. (1995) DNM1, a dynamin-related gene, participates in endosomal trafficking in yeast. J Cell Biol 130: 553-566. PubMed
Gammie AE, Tolmasky ME, Crosa JH. (1993) Functional characterization of a replication initiator protein. J Bacteriol 175: 3563-3569. PubMed
Tolmasky ME, Gammie AE, Crosa JH. (1992) Characterization of the recA gene of Vibrio anguillarum. Gene 110: 41-48. PubMed
Gammie AE, Crosa JH. (1991) Roles of DNA adenine methylation in controlling replication of the REPI replicon of plasmid pColV-K30. Mol Microbiol 5: 495-503. PubMed
Gammie AE, Crosa JH. (1991) Co-operative autoregulation of a replication protein gene. Mol Microbiol 5: 3015-3023. PubMed
Perez-Casal JF, Gammie AE, Crosa JH. (1989). Nucleotide sequence analysis and expression of the minimum REPI replication region and incompatibility determinants of pColV-K30. J Bacteriol 171: 2195-2201. PubMed
Gammie AE, Ruben LN. (1986) The phylogeny of macrophage function: antigen uptake and degradation by peritoneal exudate cells of two amphibian species and CAF1 mice. Cell Immunol 100: 577-583. PubMed