Biochemical caracterization of patient-derived MSH2 variants and their impact on hereditary nonpolyposis colorectal cancer syndrome

master's thesis

• Title (croatian): Biokemijska karakterizacija MSH2 varijanti dobivenih od pacijenata i njihov utjecaj na nasljedni nepolipozni kolorektalni tumorni sindrom
• Author: Denis Đermadi Bebek
• Mentor: Reno Hraščan (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2009-03-10, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Hereditary nonpolyposis colorectal cancer (HNPCC) syndrome accounts for about 3-5% of all colorectal cancers. This genetic disorder is associated with germline mutations in DNA mismatch repair (MMR) genes, mostly MLH1, MSH2, and MSH6. Inherited missense mutations, however, complicate the diagnostics because they do not always cause unambiguous predisposition to cancer. This leads to variable and contradictory interpretations of their pathogenicity. MSH2 is the second most frequently mutated HNPCC susceptibility locus and pathogenic MSH2 missense mutations have been shown to typically impair the repair activity of the protein. In this study, the aim was to confirm pathogenicity and furthermore clarify mechanisms of pathogenicity of 18 patient-derived MSH2 missense mutants. We also took advantage of MSH2 crystal structure and attempted to correlate the mismatch binding and ATP-catalyzed mismatch release activities with their locations. We observed that the MMR-deficient mutations situated in the amino-terminal connector and lever domains of MSH2 affected protein stability, whereas mutations in the ATPase domain mainly caused defects in mismatch binding or release. Of the MMR-proficient variants, four showed slightly reduced mismatch binding and/or release efficiences compared to wild-type protein, while two variants showed no defects in the assays. In conclusion, pathogenic missense mutations in MSH2 may interfere with different mechanisms that tend to cluster in separate protein domains with varying effects on protein stability.

Abstract (croatian)

Nasljedni nepolipozni kolorektalni tumorni (HNPCC) sindrom uzrokuje otprilike 3-5% svih kolorektalnih tumora. Ovaj genetički poremećaj povezan je sa zametnim mutacijama u genima za DNA mismatch popravak (MMR), uglavnom MLH1, MSH2, i MSH6 geni. Nasljedne missense mutacije kompliciraju dijagnozu jer ne uzrokuju uvijek jednaku predispoziciju za razvoj tumora. Upravo je to razlog kontradiktornih interpretacija njihove patogenosti. MSH2 je drugi najčešće pogođen HNPCC susceptibilni lokus. Pokazalo se da patogene MSH2 missense mutacije narušavaju aktivnost proteina pri procesu popravljanja DNA. Cilj nam je bio potvrditi patogenost, a potom razjasniti mehanizam patogenosti 18 MSH2 missense mutiranih proteina dobivenih od pacijenata. U studiji smo koristili također kristaliziranu strukturu MSH2 te pokušali povezati svojstva protein kao što su: vezivanje proteina za mismatch i ATP-katalizirano otpuštanje s lokacijama mutacija. Uočeno je kako MMR-mutacije smještene u amino terminalnoj poveznoj i lever (eng. poluga) domeni MSH2 uzrokuju pomanjkanje stabilnosti protein, dok mutacije u ATPaznoj domeni uglavnom uzrokuju probleme pri vezivanju za mismatch ili pri otpuštanju od istog. Četiri MMR proteinske varijante koje su bile uspješne u MMR-u pokazale su blago reducirano vezanje i/ili otpuštanje od mismatch-a uspoređujući s efikasnošću divljeg tipa, dok dvije varijante nisu uopće imale smanjenu efikasnost u usporedbi s divljim tipom. Kao zaključak, patogene missense mutacije u MSH2 mogu interferirati s različitim mehanizmima koji su pak povezani s različitim proteinskim domenama te tako imati različit efekt na stabilnost protein.

• Keywords: MutSα; MSH2; HNPCC; DNA repair
• Keywords (english): MutSα; MSH2; HNPCC; DNA repair
• Language: english
• URN:NBN: urn:nbn:hr:159:252778
• Study programme: Title: Biotechnology; specializations in: Biochemical-Microbiology; Course: Biochemical-Microbiology; Study programme type: university; Study level: graduate; Academic / professional title: diplomirani inženjer biotehnologije; smjer biokemijsko-mikrobiološki (diplomirani inženjer biotehnologije; smjer biokemijsko-mikrobiološki)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-08-31 09:28:31