20.500.12556/RUL-173215 Analiza različic proteinov v procesu popravljanja neujemanja DNA z dvohibridnim sistemom kvasovke Analysis of protein variants in the DNA mismatch repair process with the yeast two-hybrid system Lynchev sindrom je avtosomno dominantno dedovan sindrom, pri katerem imajo bolniki predispozicijo za razvoj rakavih obolenj. Okvarjeni so namreč geni, ki sodelujejo v procesu popravljanja neujemanja DNA (MMR). Med najpogosteje okvarjenimi je gen MLH1. Mutacije v tem genu pogosto vodijo v nezmožnost interakcije s proteinom PMS2, zato je beleženje patogenih različic genov za MMR ključno za diagnosticiranje Lynchevega sindroma. Ena od glavnih in vivo metod za preučevanje interakcij protein-protein je dvohibridni sistem kvasovke (Y2H), ki je hkrati osrednja metoda našega magistrskega dela. Y2H deluje na principu spojitve preiskovanih proteinov z domenama transkripcijskega faktorja, sklenitev katerih, v primeru interakcije preiskovanih proteinov, aktivira poročevalski sistem. Cilj naloge je bil pripraviti knjižnico alelov za protein MLH1, ki bi potencialno lahko vodile v razvoj Lynchevega sindroma. V primeru, da z Y2H pokažemo interakcijo med preiskovanima proteinoma, mutacija najverjetneje ni patogena. V ta namen smo z metodo PCR pomnožili pet fragmentov, ki so skupaj nosili zapis za šest degeneriranih kodonov. Ustvarjeno knjižnico naključnih različic smo vnesli v celice kvasovke Saccharomyces cerevisiae. Uspešnost sestavljanja knjižnice smo preverili s sekvenciranjem po Sangerju. Ugotovili smo, da je in vitro združevanje fragmentov z metodo PCR bolj učinkovito kot njihovo sestavljanje in vivo z rekombinacijskim sistemom kvasovke. Metodo Y2H smo nato izvedli v večjem volumnu in pomnožene variabilne dele v zaporedju plazmidov sekvencirali z metodo sekvenciranja amplikonov. Z bioinformatsko analizo rezultatov smo pokazali učinkovitost selektivnih gojišč za Y2H, vendar relativno nizko pokritost knjižnice v primerjavi s teoretično velikostjo. Ugotovili smo, da ja večina patogenih mutacij na mestih kodonov 636 in 661, ki se nahajata bolj proti notranjosti proteina MLH1. Lynch syndrome is an autosomal dominant inherited disorder characterized by an increased risk of developing various types of cancer. The condition is caused by mutations in genes involved in the DNA mismatch repair (MMR) system, with MLH1 being one of the most frequently affected genes. Mutations in MLH1 often lead to impaired interaction with its protein partner PMS2, which is why accurate identification of pathogenic variants of MLH1 is essential for diagnosis. One of the key in vivo methods for studying protein-protein interactions is theyeast two-hybrid (Y2H) system, which is also the central method used in this master's thesis. Y2H is base on the fusion of the protein of interest to separate domains of a transcription factor. If the proteins interact, the transcription factor is reconstituted and activates a reporter system. The aim of this work was to construct an allele library of MLH1 that may potentially contribute to the development of Lynch syndrome. If interaction between MLH1 and PMS2 is observed using Y2H, the mutation is likely non-pathogenic. To this end, a library of random MLH1 variants was generated using six degenerate codons introduced by PCR method across five synthetic fragments. The integrity of the library assembly was verified by Sanger sequencing, where we found that in vitro PCR-based assembly was more effective than in vivo recombination of fragments. Following the optimization of transformation protocol, we scaled up the Y2H assay in liquid media, isolated plasmids, amplified the variable region and submitted samples for amplicon sequencing. A subsequent bioinformatic analysis confirmed the effectiveness of the selection strategy, but also revealed that the actual diversity of the library was much lower than its theoretical potential. We found that most pathogenic mutations were located at codons 636 and 661 that are situated deeper within the gene structure. Lynchev sindrom MLH1 PMS2 Y2H Lynch syndrome MLH1 PMS2 Y2H true false false [L. Kojić] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-09-14 07:16:30 2025-09-14 07:16:36 2025-11-06 03:53:39 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 579.61:616-006(043.2) 264311 248876547 51403.pdf 51403.pdf 1 4025650290FC1DB25ECC92A07B42D294 75dea394162418f43b62ecb8df87edca3f71dbff4a28cad618fefe3e8bc6cb86 d1ae532f-9129-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=217388 Biotehniška fakulteta 0 0 0