Characterization of C- and N-terminal domains of Aquifex aeolicus MutL endonuclease: N-terminal domain stimulates the endonuclease activity of C-terminal domain in a zinc-dependent manner

Abstract

DNA MMR (mismatch repair) is an excision repair system that removes mismatched bases generated primarily by failure of the 3′–5′ proofreading activity associated with replicative DNA polymerases. MutL proteins homologous to human PMS2 are the endonucleases that introduce the entry point of the excision reaction. Deficiency in PMS2 function is one of the major etiologies of hereditary non-polyposis colorectal cancers in humans. Although recent studies revealed that the CTD (C-terminal domain) of MutL harbours weak endonuclease activity, the regulatory mechanism of this activity remains unknown. In this paper, we characterize in detail the CTD and NTD (N-terminal domain) of aqMutL (Aquifex aeolicus MutL). On the one hand, CTD existed as a dimer in solution and showed weak DNA-binding and Mn2+-dependent endonuclease activities. On the other hand, NTD was monomeric and exhibited a relatively strong DNA-binding activity. It was also clarified that NTD promotes the endonuclease activity of CTD. NTD-mediated activation of CTD was abolished by depletion of the zinc-ion from the reaction mixture or by the substitution of the zinc-binding cysteine residue in CTD with an alanine. On the basis of these results, we propose a model for the intramolecular regulatory mechanism of MutL endonuclease activity.