The predicted truncation from a cancer‐associated variant of the <i>MSH2</i> initiation codon alters activity of the MSH2‐MSH6 mismatch repair complex

Cyr, Jennifer L.; Brown, Graham D.; Stroop, Jennifer; Heinen, Christopher D.

doi:10.1002/mc.20838

Cited by 15 publications

(13 citation statements)

References 40 publications

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“…The connector domain's hydrophobic core, comprising three beta sheets and inward-facing residues of the surrounding helices, was also largely functionally intolerant to substitutions. Conversely, despite its name, the N-terminal mismatch binding domain (residues 1-124) was largely devoid of missense LOF variants, consistent with its dispensability in yeast 67 , and observations that MSH2 start codon loss is not strongly pathogenic in humans 68,69 . By contrast, prokaryotic mutS homodimerizes, with both N termini making extensive DNA contacts essential for mismatch recognition 70 .…”

Section: Functional Constraint Highlights Structural Featuresmentioning

confidence: 53%

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Jia

Burugula

Chen

et al. 2020

Preprint

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Introduction 3The lack of functional evidence for the majority of missense variants limits their clinical interpretability, 4 and poses a key barrier to the broad utility of carrier screening. In Lynch Syndrome (LS), one of the most 5 highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed 6 'variants of uncertain significance' (VUS). To systematically resolve their functional status, we performed 7 a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA 8 mismatch repair factor MSH2. The resulting sequence-function map is substantially complete, covering 9 94% of the 17,746 possible variants, and is highly concordant (≥96%) with existing functional data and 10 expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, 11 perhaps unexpectedly in view of its evolutionary conservation. These data provide ready-to-use 12 functional evidence to resolve the ~1,300 extant missense VUSs in MSH2, and may facilitate the 13 prospective classification of newly discovered variants in the clinic. 14 15 Main text 16 17 Lynch Syndrome (LS, OMIM:120435), the first discovered familial cancer syndrome 1 , confers 18 predisposition to colorectal and endometrial cancers due to the loss of DNA mismatch repair (MMR) and 19 resulting mutagenic burden. Most affected individuals inherit a single loss-of-function variant in one of 20 four MMR factors (MSH2, MLH1, 21 MSH6, PMS2), followed by a somatic 22 'second hit' inactivating the remaining 23 functional copy. Due to high 24 population prevalence (≥1:300) 2,3 , 25 clear genetic etiology, and potential 26 for prevention through intensified 27 surveillance, MMR gene variants are 28 considered clinically actionable 4 . 29 30 Missense changes together comprise 31 20-30% of LS variants 5 , and are 32 particularly challenging to interpret: 33 their functional impacts may range 34 from minimal to profound, most are 35 individually rare, and they exhibit 36 incomplete penetrance 6 . 37 Consequently, the overwhelming 38 majority of LS gene missense variants 39 listed in ClinVar (4,762/5,473, 87.0%) 40 are deemed 'variants of uncertain 41 significance', or VUS 42( Supplementary Fig.1), and cannot 43 be used to guide diagnosis. 45To address these challenges, we 46 performed a deep mutational scan 7 to 47 systematically measure the functional 48 impact of missense variants in the 49 major Lynch Syndrome gene MSH2. 50We established a human cell system 51 to model MSH2 variant function using 52 the mismatch repair proficient 8 cell 53 line HAP1 (Fig. 1a,c). First, to disrupt MMR, we derived clonal MSH2 knockout cells bearing a 19-bp frameshift deletion in MSH2 exon 6, and 55 verified that it lacked detectable MSH2 protein expression ( Supplementary Fig. 2). To restore MMR, we 56 stably reintroduced into cells either wild-type MSH2 cDNA (KO+WT) or a pathogenic founder allele 57 (KO+A636P) on inducible lentiviral constructs. After inducing expression with doxycycl...

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Section: Functional Constraint Highlights Structural Featuresmentioning

confidence: 53%

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Jia

Burugula

Chen

et al. 2020

Preprint

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“…Over time a defect could manifest a significant mutator phenotype, as has been suggested for the deletion of the N-terminus of Msh2. 68–69 Of note is the observation that a S656P change in yMsh2 (analogous to S760 in yMsh3, adjacent to the residues we tested) leads to defective Msh2-Msh6-mediated MMR and a dominant negative phenotype, indicating that it blocks the function of wild-type Msh2-Msh6. 11 Notably, msh2S656P was largely unaffected in 3’ NHTR (an Msh2-Msh3-specific activity) similar to our observations with mutations at Msh3 R761 and N762.…”

Section: Discussionmentioning

confidence: 92%

Distinct Requirements within the Msh3 Nucleotide Binding Pocket for Mismatch and Double-Strand Break Repair

Kumar¹,

Williams²,

Havens³

et al. 2013

Journal of Molecular Biology

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In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3’ non-homologous tail removal (3’NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3’ NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3’NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3’NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3’ NHTR.

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“…Recombinant UL12, MSH2-MSH3, and MSH2-MSH6 were expressed and purified from insect cells as previously described (9,53). Proteins were denatured and separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes.…”

Section: Methodsmentioning

confidence: 99%

DNA Mismatch Repair Proteins Are Required for Efficient Herpes Simplex Virus 1 Replication

Mohni

Mastrocola

Bai

et al. 2011

J Virol

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Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of its human host cell and is known to interact with many cellular DNA repair proteins. In this study, we examined the role of cellular mismatch repair (MMR) proteins in the virus life cycle. Both MSH2 and MLH1 are required for efficient replication of HSV-1 in normal human cells and are localized to viral replication compartments. In addition, a previously reported interaction between MSH6 and ICP8 was confirmed by coimmunoprecipitation and extended to show that UL12 is also present in this complex. We also report for the first time that MLH1 associates with ND10 nuclear bodies and that like other ND10 proteins, MLH1 is recruited to the incoming genome. Knockdown of MLH1 inhibits immediate-early viral gene expression. MSH2, on the other hand, which is generally thought to play a role in mismatch repair at a step prior to that of MLH1, is not recruited to incoming genomes and appears to act at a later step in the viral life cycle. Silencing of MSH2 appears to inhibit early gene expression. Thus, both MLH1 and MSH2 are required but appear to participate in distinct events in the virus life cycle. The observation that MLH1 plays an earlier role in HSV-1 infection than does MSH2 is surprising and may indicate a novel function for MLH1 distinct from its known MSH2-dependent role in mismatch repair.Herpes simplex virus 1 (HSV-1) is a large double-stranded DNA virus that replicates in the nucleus of the host cell. Although cis-and trans-acting functions involved in HSV-1 replication in infected cells have been identified (69), the mechanism of HSV-1 DNA replication remains poorly understood. The appearance of viral genomes in the nucleus might be expected to induce a cellular DNA damage response, and it is now clear that HSV-1 has evolved a complex relationship with the host DNA damage response pathways (33,48,60,66,70,71). During infection, cellular factors that are beneficial to the virus are hijacked and recruited, while other factors and pathways are degraded or inactivated. Previous work from our group and others has shown that the ATR (ATM-and Rad3-related) and DNA-PK (DNA-dependent protein kinase) DNA damage-sensing kinases are attenuated in HSV-1-infected cells, while the ATM (ataxia-telangiectasia-mutated) kinase is activated (31,34,48,50). In addition, other components of the DNA damage repair and response pathways also appear to be beneficial to the virus and are utilized during viral replication (35). Many host cellular DNA damage proteins are known to localize to viral replication compartments (33,48,60,66,71). Furthermore, in a recent proteomic analysis of replication compartments, several DNA repair proteins were identified as potential interacting partners of the viral single-stranded DNA (ssDNA) binding protein ICP8 (66). One such potential interacting partner, the DNA mismatch repair (MMR) protein MSH2, was shown to localize to the replication compartments in infected cells (66).The DNA MMR system is highly c...

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The predicted truncation from a cancer‐associated variant of the MSH2 initiation codon alters activity of the MSH2‐MSH6 mismatch repair complex

Cited by 15 publications

References 40 publications

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Distinct Requirements within the Msh3 Nucleotide Binding Pocket for Mismatch and Double-Strand Break Repair

DNA Mismatch Repair Proteins Are Required for Efficient Herpes Simplex Virus 1 Replication

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