Linker domain function predicts pathogenic MLH1 missense variants

London, James A.; Martín-López, Juana; Yang, Inho; Liu, Jiaquan; Lee, Jong–Bong; Fishel, Richard

doi:10.1073/pnas.2019215118

Cited by 14 publications

(32 citation statements)

References 24 publications

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“…1c, black dots). These observations are consistent with previous work and suggests that any singular interactions between MutL and DNA is either non-existent or significantly shorter than the imaging frame-rate (100 msec) at physiological ionic strength 18,33 . Interestingly, once on the DNA the lifetime of MutL remained constant, and the diffusion coefficient (D) did not change significantly over the range of low ionic strength conditions (Fig.…”

Section: The Mutl Ntd Positively Charged Cleft Is Indispensable For Mmrsupporting

confidence: 92%

“…2a). These observations mimic previous results showing that mismatch recognition triggers the formation of dynamic ATPbound MutS sliding clamps on DNA [22][23][24][25][26][27][28]33,61 .…”

Section: Dna Is Essential For the Initial Muts-mutl Interactionsupporting

confidence: 90%

“…We exploited prism-based single molecule total internal reflection fluorescence (smTIRF) microscopy to visualize E.coli MutL PCC in real time 18,20,33 . Single 18.4-kb DNA molecules containing a G/T mismatch were stretched across a passivated custom-made flow cell surface by laminar flow (64% of full length) and linked at both ends via biotin-neutravidin (Supplementary (which was not certified by peer review) is the author/funder.…”

Section: The Mutl Ntd Positively Charged Cleft Is Indispensable For Mmrmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted October 23, 2021. ; https://doi.org/10.1101/2021.10. 22.465456 doi: bioRxiv preprint proteins remain continuously dynamic on the DNA during MMR 18,20,33,36 . Thus, the biological function, if any, of the MutL PCC remains an enigma.…”

Section: Introductionmentioning

confidence: 99%

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MutS and DNA Function as a Clamp Loader for the MutL Sliding Clamp During Mismatch Repair

Yang

Han

et al. 2021

Preprint

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DNA mismatch repair (MMR) is accomplished by highly conserved MutS and MutL homologs. MutS proteins recognize mismatch nucleotides and in the presence of ATP form a stable sliding clamp on the DNA. The MutS sliding clamp then promotes the cascade assembly of a MutL sliding clamp, which ultimately coordinates downstream mismatch excision. The MutS clamp-loader mechanics are unknown. Here we have examined a conserved positively charged cleft (PCC) located on the MutL N-terminal domain (NTD) proposed to mediate stable DNA binding events in several MMR models. We show that MutL does not bind DNA in physiological ionic conditions. Instead, the MutS sliding clamps and DNA together exploit the PCC to position the MutL NTD for clamp loading. Once in a sliding clamp form, the MutL PCC aids in UvrD helicase capture but not interactions with MutH during mismatch excision. The MutS-DNA clamp-loader progressions are significantly different from the replication clamp-loaders that attach polymerase processivity factors such as beta-clamp and PCNA to the DNA. These studies underlining the breadth of mechanisms for stably linking crucial genome maintenance proteins to the DNA.

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Section: The Mutl Ntd Positively Charged Cleft Is Indispensable For Mmrsupporting

confidence: 92%

“…2a). These observations mimic previous results showing that mismatch recognition triggers the formation of dynamic ATPbound MutS sliding clamps on DNA [22][23][24][25][26][27][28]33,61 .…”

Section: Dna Is Essential For the Initial Muts-mutl Interactionsupporting

confidence: 90%

Section: The Mutl Ntd Positively Charged Cleft Is Indispensable For Mmrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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MutS and DNA Function as a Clamp Loader for the MutL Sliding Clamp During Mismatch Repair

Yang

Han

et al. 2021

Preprint

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“…In E.coli, these two sliding clamps function together and/or separately to enhance EcMutH DNA association and GATC-hemimethylation incision activity (18)(19)(20) as well as EcUvrD strand specific unwindingdisplacement activity (21). The human HsMSH2-HsMSH6 and HsMLH1-HsPMS2 similarly form a cascade of sliding clamps, although the detailed downstream functions remain poorly understood (22).…”

Section: Introductionmentioning

confidence: 99%

The Formation of a Stable Sliding Clamp Discriminates MSH2-MSH3 and MSH2-MSH6 Mismatch Interaction

Britton

London

Martín-López

et al. 2021

Preprint

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MutS homologs (MSH) are highly conserved core components of DNA mismatch repair (MMR). Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Once on DNA the MSH sliding clamps provide a platform for the assembly of MMR strand-specific excision components beginning with the highly conserved MutL homologs (MLH/PMS). Previous studies with short mismatch-containing oligonucleotides revealed an MSH ATP hydrolysis (ATPase) cycle that included mismatch recognition, the formation of an ATP-bound sliding clamp and dissociation from the end of a mismatched DNA that ultimately recovers the mismatch binding conformation. We found that ATP-bound MSH complexes on blocked-end or very long DNA are extremely stable under a range of ionic conditions. These observations underpinned the development of a high-throughput fluorescence resonance energy transfer (FRET) system capable of clearly distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 activities that is suitable for chemical inhibitor screens.

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

Putnam

Kolodner

2023

BioEssays

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MutL family proteins contain an N‐terminal ATPase domain (NTD), an unstructured interdomain linker, and a C‐terminal domain (CTD), which mediates constitutive dimerization between subunits and often contains an endonuclease active site. Most MutL homologs direct strand‐specific DNA mismatch repair by cleaving the error‐containing daughter DNA strand. The strand cleavage reaction is poorly understood; however, the structure of the endonuclease active site is consistent with a two‐ or three‐metal ion cleavage mechanism. A motif required for this endonuclease activity is present in the unstructured linker of Mlh1 and is conserved in all eukaryotic Mlh1 proteins, except those from metamonads, which also lack the almost absolutely conserved Mlh1 C‐terminal phenylalanine‐glutamate‐arginine‐cysteine (FERC) sequence. We hypothesize that the cysteine in the FERC sequence is autoinhibitory, as it sequesters the active site. We further hypothesize that the evolutionary co‐occurrence of the conserved linker motif with the FERC sequence indicates a functional interaction, possibly by linker motif‐mediated displacement of the inhibitory cysteine. This role is consistent with available data for interactions between the linker motif with DNA and the CTDs in the vicinity of the active site.

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Linker domain function predicts pathogenic MLH1 missense variants

Cited by 14 publications

References 24 publications

MutS and DNA Function as a Clamp Loader for the MutL Sliding Clamp During Mismatch Repair

MutS and DNA Function as a Clamp Loader for the MutL Sliding Clamp During Mismatch Repair

The Formation of a Stable Sliding Clamp Discriminates MSH2-MSH3 and MSH2-MSH6 Mismatch Interaction

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

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