The hMSH2(M688R) Lynch syndrome mutation may function as a dominant negative

Martín-López, Juana V.; Barrios, Ysamar; Medina-Arana, Vicente; Andújar, Miguel; Lee, Grace Sanghee; Gu, Liya; Li, Guo Min; Rüschoff, Josef; Salido, Eduardo; Fishel, Richard

doi:10.1093/carcin/bgs199

Cited by 9 publications

(4 citation statements)

References 48 publications

(99 reference statements)

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“…In all cases the rate appeared similar with the exception of the AF647-labeled EcMutS-his 6 /ald 6 , which appeared to form a sliding clamp approximately 2-fold better than the unlabeled EcMutS-his 6 /ald 6 . All the kinetic rate constants of EcMutS-his 6 /ald 6 binding and dissociation reported here are similar to the values obtained for EcMutS (without ald 6 -tag) in previous studies 32 47 .…”

Section: Resultssupporting

confidence: 90%

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore

Liu

Hanne

Britton

et al. 2015

Sci Rep

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Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.

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Section: Resultssupporting

confidence: 90%

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore

Liu

Hanne

Britton

et al. 2015

Sci Rep

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“…LS patients (10)(11)(12)(13). The functional studies generally investigate the consequences of MMR VUS using assays based on ectopic expression of mutant MMR genes in MMR-deficient yeast, bacteria, or human cells or on in vitro-reconstituted MMR reactions (14)(15)(16)(17)(18)(19)(20)(21)(22)(23). A possible caveat regarding such studies is that they often are performed in distantly related species, and the effect of the mutations may be masked by the unstable genetic background of MMR-deficient cells or over/under-representation of the ectopically expressed or in vitro-studied protein (9).…”

Section: Significancementioning

confidence: 99%

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Houlleberghs

Dekker

Lantermans

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that "oligo targeting" can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors.Lynch syndrome | DNA mismatch repair | MSH2 | variants of uncertain significance | site-directed mutagenesis T he DNA mismatch repair (MMR) system is essential for genome fidelity: It corrects mismatches that may arise during erroneous DNA replication and induces apoptosis if adducts caused by certain DNA-damaging agents cannot be repaired. Newly replicated DNA is scanned for base-base mispairings and loops of unpaired bases by heterodimers composed of MMR proteins mutator S homolog 2 and 6 (MSH2/MSH6) or mutator S homolog 2 and 3 (MSH2/MSH3), respectively. Upon encountering a mismatch, the MSH heterodimers recruit another heterodimer composed of mutator L homolog 1 and postmeiotic segregation increased 2 (MLH1/PMS2) to coordinate downstream repair events (1, 2). Exposure to certain DNA-damaging agents, such as methylating agents and the nucleotide analog 6-thioguanine (6TG), creates lesions in the genome that give rise to mismatches when replicated (3, 4). The DNA MMR system recognizes these mismatches and induces cell death to remove them. Several models propose how DNA MMR activates cell death. One model suggests that DNA MMR recognizes the mismatch and repetitively removes the incorporated nucleotide rather than the lesion itself, creating a cycle of futile repair which ultimately leads to DNA breakage and cell death (3). Another possibility is that MSH2/MSH6 and MLH1/PMS2 bind at the site of the damaged base and act as molecular scaffolds that activate downstream DNA damage-response pathways that result in apoptosis (5, 6). In the absence of a functional DNA MMR system, cells have an increased rate of spontaneous mutagenesis and elevated resistance to DNA-methyla...

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“…A major difference with MSH2-MSH6 is the role of MSH2-MSH3 in TNR expansion that leads to devastating diseases such as Huntington's chorea and myotonic dystrophy (41). While mutation of the core MMR components that include MSH2-MSH6 results in expansions and contractions of short simple repeats (microsatellite instability) (42,43), deletion of MSH3 virtually eliminates TNR expansion (44,45).…”

Section: Discussionmentioning

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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The hMSH2(M688R) Lynch syndrome mutation may function as a dominant negative

Cited by 9 publications

References 48 publications

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

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

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