Unexpected moves: a conformational change in MutSα enables high-affinity DNA mismatch binding.

The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a 'safety lock' that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic model explains the hypomorphic phenotype of the V63E patient mutation and other variants in the MBD interface.

Insert L1 is a central hub for allosteric regulation of USP1 activity.

During DNA replication, the deubiquitinating enzyme USP1 limits the recruitment of translesion polymerases by removing ubiquitin marks from PCNA to allow specific regulation of the translesion synthesis (TLS) pathway. USP1 activity depends on an allosteric activator, UAF1, and this is tightly controlled. In comparison to paralogs USP12 and USP46, USP1 contains three defined inserts and lacks the second WDR20-mediated activation step. Here we show how inserts L1 and L3 together limit intrinsic USP1 activity and how this is relieved by UAF1. Intriguingly, insert L1 also conveys substrate-dependent increase in USP1 activity through DNA and PCNA interactions, in a process that is independent of UAF1-mediated activation. This study establishes insert L1 as an important regulatory hub within USP1 necessary for both substrate-mediated activity enhancement and allosteric activation upon UAF1 binding.