ABSTRACT
BACKGROUND: The target substrates of DNA mismatch recognising factors MutSalpha (MSH2+MSH6) and MutSbeta (MSH2+MSH3) have already been widely researched. However, the extent of their functional redundancy and clinical substance remains unclear. Mismatch repair (MMR)-deficient tumours are strongly associated with microsatellite instability (MSI) and the degree and type of MSI seem to be dependent on the MMR gene affected, and is linked to its substrate specificities. Deficiency in MSH2 and MSH6 is associated with both mononucleotide and dinucleotide repeat instability. Although no pathogenic MSH3 mutations have been reported, its deficiency is also suggested to cause low dinucleotide repeat instability. METHODS: To assess the substrate specificities and functionality of MutSalpha and MutSbeta we performed an in vitro MMR assay using three substrate constructs, GT mismatch, 1 and 2 nucleotide insertion/deletion loops (IDLs) in three different cell lines. RESULTS: Our results show that though MutSalpha alone seems to be responsible for GT and IDL1 repair, MutSalpha and MutSbeta indeed have functional redundancy in IDL2 repair and in contrast with earlier studies, MutSbeta seems to exceed MutSalpha. CONCLUSION: The finding is clinically relevant because the strong role of MutSbeta in IDL2 repair indicates MSH3 deficiency in tumours with low dinucleotide and no mononucleotide repeat instability.
Subject(s)
DNA Mismatch Repair/genetics , DNA-Binding Proteins/physiology , Dinucleotide Repeats/genetics , MutS Homolog 2 Protein/physiology , Nucleic Acid Conformation , Animals , Cells, Cultured , DNA-Binding Proteins/genetics , HCT116 Cells , HeLa Cells , Humans , INDEL Mutation/genetics , MutS Homolog 2 Protein/genetics , MutS Homolog 3 Protein , Mutation, Missense/physiology , Spodoptera , Substrate SpecificityABSTRACT
Mismatch repair (MMR) deficiency in tumours from patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is mainly caused by mutations in the MLH1, MSH2, and MSH6 genes. A major challenge in the clinical management of patients with suspected HNPCC is the frequent occurrence of missense mutations in MSH6. These can be considered neither deleterious nor clinically innocent a priori. To assess their significance we studied five novel MSH6 missense mutations in six patients derived from a series of consecutive endometrial and colorectal cancer patients selected for study after their tumours were determined to be microsatellite unstable. We tested each mutated protein for heterodimerisation with MSH2 and for in vitro MMR capability. Four mutations (R128L, P623L, K728T, G881K+S) showed no impairment of these functions while the fifth (E1193K) displayed marked impairment of both functions. These results, taken together with our previous similar findings concerning six other missense mutations in MSH6, allow us to conclude that many or most missense changes in MSH6 likely are clinically innocent, whereas some missense changes such as E1193K, which lead to impaired MMR, are likely to be clinically significant, but have low penetrance.
