Characterization of Pathogenic Human MSH2 Missense Mutations Using Yeast as a Model System: A Laboratory Course in Molecular Biology

This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.

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Functional consequences of DNA mismatch repair missense mutations in murine models and their impact on cancer predisposition

Biochemical Society Transactions ◽

10.1042/bst0330689 ◽

2005 ◽

Vol 33 (4) ◽

pp. 689-693 ◽

Cited By ~ 9

Author(s):

S.J. Scherer ◽

E. Avdievich ◽

W. Edelmann

Keyword(s):

Mismatch Repair ◽

Genome Stability ◽

Dna Mismatch Repair ◽

Significant Proportion ◽

Missense Mutations ◽

Dna Mismatch ◽

Replication Errors ◽

Tumour Suppression ◽

Functional Consequences ◽

Dna Replication Errors

Mutations in MMR (DNA mismatch repair) genes underlie HNPCC (hereditary non-polyposis colon cancer) and also a significant proportion of sporadic colorectal cancers. MMR maintains genome stability and suppresses tumour formation by correcting DNA replication errors and by mediating an apoptotic response to DNA damage. Analysis of mouse lines with MMR missense mutations demonstrates that these MMR functions can be separated and allows the assessment of their individual roles in tumour suppression. These studies in mice indicate that, although the increased mutation rates caused by MMR defects are sufficient to drive tumorigenesis, both functions co-operate in tumour suppression.

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Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520813113 ◽

2016 ◽

Vol 113 (15) ◽

pp. 4128-4133 ◽

Cited By ~ 19

Author(s):

Hellen Houlleberghs ◽

Marleen Dekker ◽

Hildo Lantermans ◽

Roos Kleinendorst ◽

Hendrikus Jan Dubbink ◽

...

Keyword(s):

Lynch Syndrome ◽

Mismatch Repair ◽

Dna Mismatch Repair ◽

Embryonic Stem ◽

Mismatch Repair Gene ◽

Missense Mutations ◽

Repair Gene ◽

Dna Mismatch ◽

Mmr Genes ◽

Pathogenic Variants

Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that “oligo targeting” can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors.

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Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain

Nucleic Acids Research ◽

10.1093/nar/gkh855 ◽

2004 ◽

Vol 32 (18) ◽

pp. 5321-5338 ◽

Cited By ~ 27

Author(s):

A. R. Ellison

Keyword(s):

Mismatch Repair ◽

Dna Mismatch Repair ◽

Missense Mutations ◽

Atpase Domain ◽

Dna Mismatch

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Modulating Pluripotency Network Genes with Omega-3 DHA is followed by Caspase- 3 Activation and Apoptosis in DNA Mismatch Repair-Deficient/KRAS-Mutant Colorectal Cancer Stem-Like Cells

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200302113722 ◽

2020 ◽

Vol 20 (10) ◽

pp. 1221-1232

Author(s):

Nazila Mahmoudi ◽

Nowruz Delirezh ◽

Mohammad Reza Sam

Keyword(s):

Colorectal Cancer ◽

Mismatch Repair ◽

Caspase 3 ◽

Dna Mismatch Repair ◽

Cell Number ◽

Molecular Techniques ◽

Omega 3 ◽

Dna Mismatch ◽

Colorectal Cancer Stem Cells

Background: Targeting DNA mismatch repair-deficient/KRAS-mutant Colorectal Cancer Stem Cells (CRCSCs) with chemical compounds remains challenging. Modulating stemness factors Bmi-1, Sox-2, Oct-4 and Nanog in CRCSCs which are direct downstream targets of carcinogenesis pathways may lead to the reactivation of caspase-3 and apoptosis in these cells. Omega-3 DHA modulates different signaling pathways involved in carcinogenesis. However, little is known, whether in vitro concentrations of DHA equal to human plasma levels are able to modulate pluripotency genes expression, caspase-3 reactivation and apoptosis in DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells. Methods: DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells (LS174T cells) were treated with DHA, after which, cell number and proliferation-rate, Bmi-1, Sox-2, Nanog and Oct-4 expression, caspase-3 activation and apoptosis were evaluated with different cellular and molecular techniques. Results: DHA changed the morphology of cells to apoptotic forms and disrupted cell connections. After 48h treatment with 50- to 200μM DHA, cell numbers and proliferation-rates were measured to be 86%-35% and 93.6%-45.7% respectively. Treatment with 200 μM DHA dramatically decreased the expression of Bmi-1, Sox- 2, Oct-4 and Nanog by 69%, 70%, 97.5% and 53% respectively. Concurrently, DHA induced caspase-3 activation by 1.8-4.7-fold increases compared to untreated cells. An increase in the number of apoptotic cells ranging from 9.3%-38.4% was also observed with increasing DHA concentrations. Conclusion: DHA decreases the high expression level of pluripotency network genes suggesting Bmi-1, Sox-2, Oct-4 and Nanog as promising molecular targets of DHA. DHA reactivates caspase-3 and apoptosis in DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells, representing the high potential of this safe compound for therapeutic application in CRC.

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