The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

Abstract

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair

“…Thus, our studies provide mechanistic support for the idea that IDRs license Mlh1-Pms1 to interact stably interact with DNA (Fig 5) during MMR. It complements previous studies showing that IDRs regulate how a DNA repair enzyme scans chromatin for DNA lesions and how repair functions (for example, nicking of the newly replicated strand during MMR) are activated (Gorman et al , 2012; Kim et al , 2019; Gorman et al , 2010; Liu et al , 2016; Mardenborough et al , 2019).…”

“…The above models argue that the MLH IDRs play key roles in facilitating these steps, but it remains unclear how they occur (Kim et al , 2019; Sacho et al , 2008, Mardenborough et al , 2019). Sacho et al (2008) and Kunkel and Erie (2015) proposed that ATP binding to the Mlh1 subunit of the Mlh1-Pms1 heterodimer promotes the formation of a one-armed collapsed complex in which the N-terminal ATP binding domain of Mlh1 and its IDR fold into or near the C-terminal Mlh1-Pms1 domain (Fig 1B).…”

Mismatch Repair: From Preserving Genome Stability to Enabling Mutation Studies in Real-Time Single Cells

“…The alternative molecular switch model, which better fits recent E. coli single-molecule data, postulates that MutS conformational changes induced by ATP binding allow the protein to slide along the DNA passively [ 104 , 130 , 132 ]. Both active translocation and molecular switch models are consistent with the observation that a protein roadblock placed on DNA between a mismatch and an incision site can inhibit the repair substantially [ 133 , 134 , 135 ]. Finally, remote signaling may not be accomplished by a single mechanism, and both the stationary and mobile models might have their own advantages.…”

Linker domain function predicts pathogenic MLH1 missense variants

“…2 B and SI Appendix , Extended Methods ). Increasing linker domain deletions of the EcMutL and Sacchromyces cerevisae (Sc) ScMlh1 or ScPms1 (the Sc homolog of HsPMS2) initially prevents their ability to transit roadblocks on the DNA and eventually completely inhibits MMR ( 22 , 23 ). The most straightforward interpretation of these observations is that MLH/PMS sliding clamps mimic a very flexible donut that encircles the DNA within a relatively large donut hole ( Fig.…”