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

Abstract

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif ( Saccharomyces cerevisiae residues 391–415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae . These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

Insights into DNA cleavage by MutL homologs from analysis of conserved motifs in eukaryotic Mlh1

“…Reanalysis of lysine crosslinking of S. cerevisiae Mlh1-Pms1 in the absence of ATP and DNA (Figure 4A) [49] suggests that the linker motif interacts with the Mlh1-Pms1 CTDs. Several sets of crosslinks can be explained by motif-CTD interactions combined with Mlh1 CTD wrapping by the Pms1 linker as observed using in silico modeling (Figure 4B) [35] : The cladogram is based on previous analyses. [43] (B) The conserved linker motif from the alignment of selected Mlh1 sequences; yellow highlighted residues are conserved and red highlighted residues are at −0/−2 sequence difference.…”

“…Bars above indicate mutations causing full (purple), partial (green) or no (blue) MMR defects. [23,35,60,61] Vertical hashes indicate residues equivalent to DNA contacting residues in each subunit (MutL A and MutL B ) of the E. coli MutL-DNA structure. [58] Sites of S. cerevisiae Mlh1 adjacent to DNA in a Mlh1-Mlh3 bound to a Holliday junction [23] are indicated by squares.…”

Emerging Role of Plant-Based Dietary Components in Post-Translational Modifications Associated with Colorectal Cancer

“…Among the different proteins that suffer dysregulated post-translational methylation associated with CRC ( Table 2 ), the one that is involved in cell growth suppression has downregulated methylation (putative insulin-like growth factor 2 antisense gene protein) [ 44 , 45 ]. The other proteins identified have an upregulated methylation, among them are (1) BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 that is involved in apoptosis [ 46 ]; (2) homeobox protein CDX-2 that is involved in the transcriptional regulation of different genes expressed in the intestine [ 47 ]; (3) C-X-C motif chemokine 14 that is involved in immunoregulatory and inflammatory processes [ 48 ]; (4) transcription factor E2F1 that participates in the cell cycle [ 49 ]; (5) DNA mismatch repair protein Mlh1 that participates in DNA repair [ 50 ]; (6) nuclear factor NF-kappa-B p105 subunit that is a pleiotropic transcription factor involved in several signal transduction events which are initiated by stimuli such as oxidative stress or inflammation [ 51 ]; and (7) 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 that is an essential protein for cell cycle progression and apoptosis prevention [ 52 ].…”

Order out of disorder: Regulation of endonuclease activity during eukaryotic mismatch repair

“…Recent research identified an evolutionarily conserved stretch of amino acids in the linker of the yeast Mlh1 protein that is essential for endonuclease activity in vitro and MMR in vivo. 3 The paper by Put-…”

“…Recent research identified an evolutionarily conserved stretch of amino acids in the linker of the yeast Mlh1 protein that is essential for endonuclease activity in vitro and MMR in vivo 3 . The paper by Putnam and Kolodner in this issue of BioEssays 4 presents an intriguing hypothesis to explain the role of this motif in licencing endonuclease activity during eukaryotic MMR.…”