Conservation of functional asymmetry in the mammalian MutLα ATPase

Abstract

The DNA mismatch repair (MMR) protein dimer MutLα is comprised of the MutL homologues MLH1 and PMS2, which each belong to the family of GHL ATPases. These ATPases undergo functionally important conformational changes, including dimerization of the NH₂-termini associated with ATP binding and hydrolysis. Previous studies in yeast and biochemical studies with the mammalian proteins established the importance of the MutLα ATPase for overall MMR function. Additionally, the studies in yeast demonstrated a functional asymmetry between the contributions of the Mlh1 and Pms1 ATPase domains to MMR that was not reflected in the biochemical studies. We investigated the effect of mutating the highly conserved ATP hydrolysis and Mg²(+) binding residues of MLH1 and PMS2 in mammalian cell lines. Amino acid substitutions in MLH1 intended to impact either ATP binding or hydrolysis disabled MMR, as measured by instability at microsatellite sequences, to an extent similar to MLH1-null mutation. Furthermore, cells expressing these MLH1 mutations exhibited resistance to the MMR-dependent cytotoxic effect of 6-thioguanine (6-TG). In contrast, ATP hydrolysis and binding mutants of PMS2 displayed no measurable increase in microsatellite instability or resistance to 6-TG. Our findings suggest that, in vivo, the integrity of the MLH1 ATPase domain is more critical than the PMS2 ATPase domain for normal MMR functions. These in vivo results are in contrast to results obtained previously in vitro that showed no functional asymmetry within the MutLα ATPase, highlighting the differences between in vivo and in vitro systems.

Figure 1. Protein expression in MC2 and C18 parent and complemented cell lines

Western blots of MC2 (Mlh1−/−) cells expressing WT MLH1, MLH1-E34A, and MLH1-N38A in the left panel and C18 (Pms2−/−) cells expressing WT PMS2, PMS2-E41A, and PMS2-N45A in the right panel. The MSH6 signal was used as a loading control. MC2 cells display reduced PMS2 expression, as PMS2 depends on MLH1 for protein stability [23].

Figure 2. 6-Thioguanine response in MLH1- and PMS2-expressing cells

Cells were plated out and exposed to varying doses of 6-thioguanine for 24 hours. Colonies were stained with methylene blue and counted 8–10 days after plating. A) MC2, MLH1-WT, MLH1-E34A, or MLH1-N38A cells. B) C18, PMS2-WT, PMS2-E41A, or PMS2-N45A cells.