Functional Characterization of MutS Homologue Mismatch Repair Proteins and their Variants
Year: 2012
Type: dissertation
Abstract: Lynch syndrome (LS) is one of the most common hereditary cancer syndromes and may lead to cancer development, mainly in colon or in endometrium, for 20 years earlier than in general population. LS is an autosomal dominantly inherited disorder, associated with the malfunction of a highly conserved postreplicative DNA mismatch repair (MMR) mechanism and germline mutations at least in four different MMR genes, MLH1, MSH2, MSH6, and PMS2. The MMR genes MSH3 and MLH3 have also been linked to LS but their roles are less clear. To be able to offer an appropriate follow-up and genetic counseling to LS families and their mutation carriers, they must be diagnosed, which usually starts by studying the cancer history in the family and the tumor phenotype of the index patient followed by mutation search and pathogenicity assessment of found variations. The amino acid changes which change only one amino acid in a protein structure, do not necessarily destroy protein and therefore their pathogenicity is difficult to interpret. Furthermore, in rare cases, an individual can carry two variations either in the same or different MMR genes, which further complicates the pathogenicity assessment. DNA MMR corrects mismatches arising mainly during DNA replication. DNA synthesis in each cell division is carried out by three major replicative DNA polymerases (Pols) α, δ and ε and their incomplete proofreading activity together with malfunction in MMR leads to the accumulation of mismatches in the genome leading to genomic instability and cancer. MutS homologue (MSH) MMR proteins form the DNA mismatch recognizing factors MutSα (MSH2/MSH6) and MutSβ (MSH2/MSH3). One aim in the present study was to analyze the substrate efficiencies of MutSα and MutSβ by using the functional in vitro MMR assay with different substrates and cell lines. The target substrates of MutSα and MutSβ have already been widely studied. However, the extent of their functional redundancy and clinical substance remains unclear. Here, our results show that although MutSα alone seems to be responsible for the mismatch and one nucleotide loop repair, MutSα and MutSβ have functional redundancy in two nucleotide loop repair and MutSβ even seems to exceed MutSα in that. The finding is clinically relevant since such a strong role in two nucleotide loop repair indicates MSH3 deficiency in tumors with low dinucleotide and no mononucleotide repeat instability. The second aim in the study was to functionally characterize a possible compound effect of 9 pairs of variants of unknown significance (VUS) found in cancer patients. Four variant pairs were shown to be proficient while one VUS, MSH2 c.380A>G was individually assessed proficient but in a pair with another VUS deficient. Thus, our results suggest that two inherited MMR gene variations in a cancer patient may have a concomitant contribution to MMR deficiency. Moreover, the role of this frequently reported MMR gene VUS MSH2 c.380A>G is especially interesting, since its concomitant defect with another variant could finally explain its recurrent occurrence in colorectal cancer patients. Three MSH6 VUS were shown to cause MMR deficiency individually. Furthermore, one separately studied MSH3 variation was shown to be proficient in MMR. The third aim was to study the role of replicative polymerases α and ε in MMR. Here, we demonstrate a proliferating cell nuclear antigen independent interaction between replicative DNA polymerases and MSH proteins MSH2 and MSH6 by co-purification as well as by conventional and chromatin immunoprecipitation. Chromatin recruitment but not the release of MSH2 appears to depend on DNA replication. The novel interaction provides a potential mechanism for replication-dependent strand discrimination during MMR. In addition, we showed that polymerases of the replication fork have a functional role in human MMR. Our data, suggesting that MSH2 and MSH6 physically interact with Pols δ and Pol α, are in accordance with models where MSH proteins are continuously loaded onto chromatin in a replication-dependent manner and persist on DNA that has already completed replication.
Source: Työväentutkimus Vuosikirja
Author: Jukka Kantelinen
Language: English
Subfield: Pathology and Forensic Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1606410884