Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair

Abstract

Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage1. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis2. Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10–40% of related sporadic tumours3. However, the collaborative mechanics of MSH and MLH/PMS proteins have not been resolved in any organism. We visualized Escherichia coli (Ec) ensemble mismatch repair and confirmed that EcMutS mismatch recognition results in the formation of stable ATP-bound sliding clamps that randomly diffuse along the DNA with intermittent backbone contact. The EcMutS sliding clamps act as a platform to recruit EcMutL onto the mismatched DNA, forming an EcMutS–EcMutL search complex that then closely follows the DNA backbone. ATP binding by EcMutL establishes a second long-lived DNA clamp that oscillates between the principal EcMutS–EcMutL search complex and unrestricted EcMutS and EcMutL sliding clamps. The EcMutH endonuclease that targets mismatch repair excision only binds clamped EcMutL, increasing its DNA association kinetics by more than 1,000-fold. The assembly of an EcMutS–EcMutL–EcMutH search complex illustrates how sequential stable sliding clamps can modulate one-dimensional diffusion mechanics along the DNA to direct mismatch repair.

The torpedo effect inBacillus subtilis:RNase J1 resolves stalled transcription complexes

“…A number of proteins involved in DNA repair were upregulated in the mutant strain (23 genes Table EV5), which may, at least in part, stem from feedback mechanisms increasing the amount of these proteins to help the cell cope with increased number of transcription-replication collisions with mutagenic/DNA damaging effects. These upregulated genes included genes for mismatch repair mutS, mutL (Liu et al, 2016;LeBlanc et al, 2018), nucleotide excision repair pcrA (Sanders et al, 2017), base excision repair mutM, mutT, ung, processing of abasic sites yqfS, exoA, yshC (Lenhart et al, 2012), and genes for restart after replication-transcription collision addA, addB, recA (Shepanek et al, 1989;Krajewski et al, 2014). Interestingly, genes involved in DNA repair after UV damage (UvrABC; Lenhart et al, 2012) were either unchanged or upregulated.…”

Clamping down on copy errors

“…b , Liu et al 4 report that, once a mismatch is detected, MutS recruits the protein MutL, and the two proteins slowly slide together up and down DNA. c , MutL in turn recruits the enzyme MutH, and the three move as one complex.…”

“…In the bacterium Escherichia coli , repair requires communication between enzymes across long stretches of DNA, and how this occurs has been hotly debated for decades 1–3 . On page 583, Liu et al 4 help to solve this mystery by using state-of-the-art techniques to analyse mismatch repair at the single-molecule level.…”

Expression and purification of nuclease-free protocatechuate 3,4-dioxygenase for prolonged single-molecule fluorescence imaging

“…Fluorescence detection is a powerful analytical tool with sensitivity at the SM level that allows visualizing a cohort of individual biomolecules in action. SM fluorescence imaging can generate reaction trajectories of long-range movements such as sliding of an enzyme on DNA [14, 24] as well as short-range movements such as conformational transitions of RNAs or proteins [15–18, 25, 26, 28]. Direct visualization of individual reaction trajectories allows identification of the dynamics, formation of transient intermediates, stochastic and asynchronous events as underlying principles of biology.…”