The unstructured linker of Mlh1 contains a motif required for endonuclease function which is mutated in cancers

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Eukaryotic DNA mismatch repair (MMR) is crucial for maintaining genomic stability.
• The Mlh1-Pms1 endonuclease complex plays a central role in MMR by cleaving mispaired DNA.
• Both Mlh1 and Pms1 proteins possess long unstructured linkers connecting their functional domains.

Purpose of the Study:

• To identify and characterize functional motifs within the Mlh1 linker essential for MMR.
• To investigate the role of a conserved Mlh1 linker motif in the endonuclease activity and recruitment of the Mlh1-Pms1 complex.

Main Methods:

• Site-directed mutagenesis to create Mlh1 linker motif mutants (Mlh1-R401A,D403A-Pms1).
• In vitro assays to assess MMR and endonuclease activity of the mutant complex.
• In vivo functional analysis of the motif in different linker locations.
• Modeling of human cancer-associated motif mutations in Saccharomyces cerevisiae.

Main Results:

• A conserved Mlh1 linker motif (S. cerevisiae residues 391-415) was identified as critical for MMR.
• Mutating this motif abolished Mlh1-Pms1 endonuclease activity in vitro, despite the motif's distance from active sites.
• Peptides containing the motif inhibited wild-type Mlh1-Pms1 endonuclease activity.
• The motif's function was demonstrated in vivo at various linker positions, and cancer-associated mutations resulted in loss-of-function.

Conclusions:

• The Mlh1 linker motif is essential for the PCNA-activated endonuclease activity of the Mlh1-Pms1 complex.
• This motif likely facilitates MMR through interactions with DNA, PCNA, RFC, or other components of the MMR machinery.
• Understanding this motif's function provides insights into MMR mechanisms and potential therapeutic targets for MMR-deficient cancers.