Steady-state regulation of the human DNA mismatch repair system

Abstract

Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription- polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady-state levels of mRNA expression correlated well with protein expression, hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins. Similarly, hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1 restored HCT116+ch3 cells, hPMS2 protein was reexpressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5-5-fold higher than in their nontransformed counterpart. The steady-state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 protein depends upon hMH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after vital transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell.