Skip navigation
Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp012n49t172r
Full metadata record
DC FieldValueLanguage
dc.contributor.advisorRose, Marken_US
dc.contributor.advisorGammie, Alisonen_US
dc.contributor.authorArlow, Timen_US
dc.contributor.otherMolecular Biology Departmenten_US
dc.date.accessioned2012-08-01T19:33:34Z-
dc.date.available2012-08-01T19:33:34Z-
dc.date.issued2012en_US
dc.identifier.urihttp://arks.princeton.edu/ark:/88435/dsp012n49t172r-
dc.description.abstractMSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically-identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of over half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wild-type levels restored mismatch repair, suggesting that overexpression is not always required to restore function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants. We identified the ubiquitin ligase San1, known to target misfolded nuclear proteins, as being responsible for targeting the Msh2 variants. Deletion of San1 restored protein levels for 18 of the 19 variants but did not elevate wild-type Msh2 levels. The unstable variants interacted with a RING domain deficient form of San1, whereas wild-type Msh2 did not. Additionally, deletion of SAN1 suppressed the mismatch repair defect of multiple unstable variants. Of medical significance, the clinically approved drug Bortezomib (VelcadeĀ®) partially restored protein levels and mismatch repair function for low level variants and reversed the resistance to the chemotherapeutic Cisplatin. Our results provide the foundation for an innovative therapeutic regime for certain mismatch repair defective cancers that are refractory to conventional chemotherapies. Msh2 heterodimerizes with Msh6 to form the MutS alpha complex which is responsible for the recognition of DNA mismatches. In this work we show that formation of the MutS alpha heterodimer is stabilizing for both Msh2 and Msh6. Specifically, deleting MSH6 from the cell causes wild-type Msh2 to be turned over by the ubiquitin-mediated pathway resulting in lower steady-state levels of Msh2. Furthermore, only unstable Msh2 missense variants that interact with Msh6 are destabilized in the absence of Msh6; whereas those that do not interact are not further destabilized in cells lacking Msh6. Previously, we identified all but one of the clinically significant low-level missense Msh2 variants are stabilized in a strain lacking the San1 ubiquitin ligase. Here we show that the exception, Msh2-R542P, is targeted by Rad7. Deleting RAD7 fully restored Msh2-R542P levels; however, deleting CUL3, RAD16, RAD4, and ELC1 did not, suggesting a novel or redundant ligase complex. Finally, we determined that the wild-type, monomeric form of Msh2 is not targeted by either San1 or Rad7, suggesting a third ligase is involved in clearing unbound Msh2 from the cell. In summary, this work provides evidence that alternative forms of the Msh2 protein are targeted by different ubiquitin ligases.en_US
dc.language.isoenen_US
dc.publisherPrinceton, NJ : Princeton Universityen_US
dc.relation.isformatofThe Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the <a href=http://catalog.princeton.edu> library's main catalog </a>en_US
dc.subjectBortezomiben_US
dc.subjectDNA Mismatch Repairen_US
dc.subjectUbiquitinationen_US
dc.subject.classificationMolecular biologyen_US
dc.titleUbiquitination of the DNA Mismatch Repair Protein Msh2en_US
dc.typeAcademic dissertations (Ph.D.)en_US
pu.projectgrantnumber690-2143en_US
Appears in Collections:Molecular Biology

Files in This Item:
File Description SizeFormat 
Arlow_princeton_0181D_10182.pdf11.34 MBAdobe PDFView/Download


Items in Dataspace are protected by copyright, with all rights reserved, unless otherwise indicated.