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Eric Alani - One of the best experts on this subject based on the ideXlab platform.
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Expanded roles for the MutL family of DNA mismatch repair proteins
Yeast, 2020Co-Authors: Christopher M. Furman, Ryan Elbashir, Eric AlaniAbstract:The MutL family of DNA mismatch repair proteins plays a critical role in excising and repairing misincorporation errors during DNA replication. In many eukaryotes, members of this family have evolved to modulate and resolve recombination intermediates into crossovers during meiosis. In these organisms, such functions promote the accurate segregation of chromosomes during the meiosis I division. What alterations occurred in MutL homolog (MLH) family members that enabled them to acquire these new roles? In this review, we present evidence that the yeast MLH1-Mlh3 and MLH1-Mlh2 complexes have evolved novel enzymatic and nonenzymatic activities and protein-protein interactions that are critical for their meiotic functions. Curiously, even with these changes, these complexes retain backup and accessory roles in DNA mismatch repair during vegetative growth.
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a mutation in the endonuclease domain of mouse mlh3 reveals novel roles for mutlγ during crossover formation in meiotic prophase i
bioRxiv, 2019Co-Authors: Melissa Toledo, Vandana Raghavan, Xianfei Sun, Miguel A Brienoenriquez, Stephen K Gray, Jeffrey Pea, Anita Venkatesh, Lekha Patel, Peter L Borst, Eric AlaniAbstract:During meiotic prophase I, double strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse Mlh3DN allele harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have normal DSBs and undergo normal synapsis events in early prophase I. Unlike Mlh3-/- males, however, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, DSB repair and CO designation factors persist in Mlh3DN/DN males, indicating a temporal delay in repair events. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may be partially functional or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males, with evidence favoring the latter option. Double mutant mice that are homozygous for the Mlh3DN/DN mutation along with a null allele of Mus81, show losses in chiasmata approaching levels observed in Mlh3-/- males, suggesting that the MUS81-EME1-regulated crossover pathway accounts for some of the increased residual chiasmata observed in the Mlh3DN/DN spermatocytes. These results demonstrate that an intact MLH3 endonuclease domain is essential for most COs in mammalian meiosis, and that an endonuclease-impaired MutLγ retains the ability to facilitate the recruitment of other repair pathways, including MUS81-EME1.
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mlh3 mutations in baker s yeast alter meiotic recombination outcomes by increasing noncrossover events genome wide
PLOS Genetics, 2017Co-Authors: Najla Alsweel, Carol M Manhart, Jennifer A Surtees, Vandana Raghavan, Abhishek Dutta, V P Ajith, Luigi Di Vietro, Nabila Khondakar, K T Nishant, Eric AlaniAbstract:MLH1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how MLH1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. MLH1-mlh3 representatives for each class were purified and characterized. Both MLH1-mlh3-32 (MMR+, crossover-) and MLH1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with MLH1-Mlh3 in MMR, stimulated the endonuclease activity of MLH1-mlh3-32 but not MLH1-mlh3-45, suggesting that MLH1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating MLH1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.
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the mismatch repair and meiotic recombination endonuclease MLH1 mlh3 is activated by polymer formation and can cleave dna substrates in trans
PLOS Biology, 2017Co-Authors: Carol M Manhart, Martin White, Joaquin Ortega, Jennifer A Surtees, Eric AlaniAbstract:Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of MLH1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the MLH1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that MLH1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient MLH1-Mlh3. In addition, MLH1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that MLH1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which MLH1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.
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mlh3 separation of function and endonuclease defective mutants display an unexpected effect on meiotic recombination outcomes
bioRxiv, 2017Co-Authors: Najla Alsweel, Carol M Manhart, Jennifer A Surtees, Abhishek Dutta, V P Ajith, Luigi Di Vietro, Nabila Khondakar, K T Nishant, Eric AlaniAbstract:MLH1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how MLH1-Mlh3 functions in both meiosis and MMR, we analyzed in bakers yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. MLH1-mlh3 representatives for each separation of function class were purified and characterized. Both MLH1-mlh3-32 (MMR+, crossover-) and MLH1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with MLH1-Mlh3 in MMR, stimulated the endonuclease activity of MLH1-mlh3-32 but not MLH1-mlh3-45, suggesting that MLH1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for two mlh3 mutants with opposite separation of function phenotypes, and an endonuclease defective mutant. Unexpectedly, all three showed increases in the number of non-crossover events that were not observed in mlh3null. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating MLH1-Mlh3 enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.
Päivi Peltomäki - One of the best experts on this subject based on the ideXlab platform.
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Lynch Syndrome Genes
Familial Cancer, 2005Co-Authors: Päivi PeltomäkiAbstract:Since the discovery of the major human genes with DNA mismatch repair (MMR) function in 1993--1995, mutations in four, MSH2 , MLH1 , MSH6 , and PMS2 , have been convincingly linked to susceptibility of hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome. Among these, PMS2 mutations are associated with diverse clinical features, including those of the Turcot syndrome. Two additional MMR genes, MLH3 and PMS1 , have also been proposed to play a role in Lynch syndrome predisposition, but the clinical significance of mutations in these genes is less clear. According to the database maintained by the International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer (ICG-HNPCC), current InSiGHT (International Society for Gastrointestinal Hereditary Tumors), approximately 500 different HNPCC-associated MMR gene mutations are known that primarily involve MLH1 (~50%), MSH2 (~40%), and MSH6 (~10%). Examination of HNPCC/Lynch syndrome-associated MMR genes and their mutations has revealed several other important functions for their protein products beyond postreplicative mismatch repair as well as many alternative mechanisms of pathogenicity. Despite these advances, much is yet to be learned about the molecular basis of correlations between genetic changes and clinical features of the disease.
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Deficient DNA Mismatch Repair in Carcinogenesis
Handbook of experimental pharmacology, 2003Co-Authors: Päivi PeltomäkiAbstract:The postreplicative DNA mismatch repair (MMR) system maintains genome integrity by recognizing and repairing mismatched nucleotides that result from misincorporation during DNA synthesis. The genes coding for MMR proteins are highly conserved throughout evolution. The human proteins that correspond to the bacterial MutS proteins and participate in MMR include MSH2, MSH3, and MSH6, whereas the human MutL proteins include MLH1, PMS1, PMS2, and MLH3. Characteristics of the human MMR genes are given in Table 1.
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Genetic and epigenetic modification of MLH1 accounts for a major share of microsatellite-unstable colorectal cancers.
The American journal of pathology, 2000Co-Authors: Shannon A. Kuismanen, Albert De La Chapelle, Mari T. Holmberg, Reijo Salovaara, Päivi PeltomäkiAbstract:Microsatellite instability (MSI) is a hallmark of hereditary nonpolyposis colorectal cancer, and in these patients, results from inherited defects in DNA mismatch repair genes, mostly MSH2 and MLH1. MSI also occurs in 15% of sporadic colorectal cancers, but in these tumors, its basis is less well characterized. We investigated 46 sporadic MSI+ colorectal cancers for changes in MSH2 and MLH1 protein expression, followed by the analysis of somatic mutation, loss of heterozygosity (LOH), and promoter hypermethylation as possible underlying defects. Most cases (36/46, 78%) showed lost or reduced MLH1 expression. Among these, a majority (83%) was associated with MLH1 promoter hypermethylation, whereas the rates of LOH and somatic mutation of MLH1 were 24% and 13%, respectively. Hypermethylation and LOH were inversely correlated, suggesting that they had alternative functions in the inactivation of MLH1. MSH2 expression was lost in 7/46 (15%), and of these, 2 (29%) showed LOH and/or somatic mutation of MSH2. We conclude that most sporadic MSI+ colorectal cancers have an MLH1-associated etiology and that epigenetic modification is a major mechanism of MLH1 inactivation. Moreover, we found a significantly lower prevalence for MLH1 promoter hypermethylation in hereditary nonpolyposis colorectal cancer tumors with MLH1 germline mutations (12/26, 46%), which might explain some differences that are known to occur in the clinicopathological characteristics and tumorigenic pathways between sporadic and hereditary MSI+ colorectal cancers.
Robert Brown - One of the best experts on this subject based on the ideXlab platform.
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Interactions of the DNA mismatch repair proteins MLH1 and MSH2 with c-MYC and MAX
Oncogene, 2003Co-Authors: Mary Mac Partlin, Stephen T Durant, Carol Mccormick, Elizabeth Homer, Helen Robinson, Dorothy H Crouch, Elizabeth C Matheson, Andrew G Hall, David Af Gillespie, Robert BrownAbstract:MSH2 and MLH1 have a central role in correcting mismatches in DNA occurring during DNA replication and have been implicated in the engagement of apoptosis induced by a number of cytotoxic anticancer agents. The function of MLH1 is not clearly defined, although it is required for mismatch repair (MMR) and engagement of apoptosis after certain types of DNA damage. In order to identify other partners of MLH1 that may be involved in signalling MMR or apoptosis, we used human MLH1 in yeast two-hybrid screens of normal human breast and ovarian cDNA libraries. As well as known partners of MLH1 such as PMS1, MLH3 and MBD4, we identified the carboxy terminus of the human c-MYC proto-oncogene as an interacting sequence. We demonstrate, both in vitro by yeast two-hybrid and GST-fusion pull-down experiments, as well as in vivo by coimmunoprecipitation from human tumour cell extracts, that MLH1 interacts with the c-MYC protein. We further demonstrate that the heterodimeric partner of c-MYC, MAX, interacts with a different MMR protein, MSH2, both in vitro and in vivo. Using an inducible c-MYC-ER™ fusion gene, we show that elevated c-MYC expression leads to an increased HGPRT mutation rate of Rat1 cells and an increase in the number of frameshift mutants at the HGPRT locus. The effect on HGPRT mutation rate is small (2–3-fold), but is consistent with deregulated c-MYC expression partially inhibiting MMR activity.
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reversal of drug resistance in human tumor xenografts by 2 deoxy 5 azacytidine induced demethylation of the hMLH1 gene promoter
Cancer Research, 2000Co-Authors: Jane A Plumb, Gordon Strathdee, Julieann Sludden, Stanley B Kaye, Robert BrownAbstract:Loss of DNA mismatch repair because of hypermethylation of the hMLH1 gene promoter occurs at a high frequency in a number of human tumors. A role for loss of mismatch repair (MMR) in resistance to a number of clinically important anticancer drugs has been shown. We have investigated whether the demethylating agent 2′-deoxy-5-azacytidine (DAC) can be used in vivo to sensitize MMR-deficient, drug-resistant ovarian (A2780/cp70) and colon (SW48) tumor xenografts that are MLH1 negative because of gene promoter hypermethylation. Treatment of tumor-bearing mice with the demethylating agent DAC at a nontoxic dose induces MLH1 expression. Re-expression of MLH1 is associated with a decrease in hMLH1 gene promoter methylation. DAC treatment alone has no effect on the growth rate of the tumors. However, DAC treatment sensitizes the xenografts to cisplatin, carboplatin, temozolomide, and epirubicin. Sensitization is comparable with that obtained by reintroduction of the hMLH1 gene by chromosome 3 transfer. Consistent with loss of MMR having no effect on sensitivity in vitro to Taxol, DAC treatment has no effect on the Taxol sensitivity of the xenografts. DAC treatment does not sensitize xenografts of HCT116, which lacks MMR because of hMLH1 mutation. Because there is emerging data on the role of loss of MMR in clinical drug resistance, DAC could have a role in increasing the efficacy of chemotherapy for patients whose tumors lack MLH1 expression because of hMLH1 promoter methylation.
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dependence on rad52 and rad1 for anticancer drug resistance mediated by inactivation of mismatch repair genes
Current Biology, 1999Co-Authors: Stephen T Durant, Melanie M Morris, Maureen Illand, Helen J Mckay, Carol Mccormick, Gillian L Hirst, Rhona H Borts, Robert BrownAbstract:Abstract Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3,4]. We report that inactivation of MMR genes ( MLH1 , MLH2 , MSH2 , MSH3 , MSH6 , but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in MLH1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1 , MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins – in addition to their role in DNA-damage recognition – decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.
Carol M Manhart - One of the best experts on this subject based on the ideXlab platform.
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mlh3 mutations in baker s yeast alter meiotic recombination outcomes by increasing noncrossover events genome wide
PLOS Genetics, 2017Co-Authors: Najla Alsweel, Carol M Manhart, Jennifer A Surtees, Vandana Raghavan, Abhishek Dutta, V P Ajith, Luigi Di Vietro, Nabila Khondakar, K T Nishant, Eric AlaniAbstract:MLH1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how MLH1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. MLH1-mlh3 representatives for each class were purified and characterized. Both MLH1-mlh3-32 (MMR+, crossover-) and MLH1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with MLH1-Mlh3 in MMR, stimulated the endonuclease activity of MLH1-mlh3-32 but not MLH1-mlh3-45, suggesting that MLH1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating MLH1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.
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the mismatch repair and meiotic recombination endonuclease MLH1 mlh3 is activated by polymer formation and can cleave dna substrates in trans
PLOS Biology, 2017Co-Authors: Carol M Manhart, Martin White, Joaquin Ortega, Jennifer A Surtees, Eric AlaniAbstract:Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of MLH1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the MLH1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that MLH1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient MLH1-Mlh3. In addition, MLH1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that MLH1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which MLH1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.
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mlh3 separation of function and endonuclease defective mutants display an unexpected effect on meiotic recombination outcomes
bioRxiv, 2017Co-Authors: Najla Alsweel, Carol M Manhart, Jennifer A Surtees, Abhishek Dutta, V P Ajith, Luigi Di Vietro, Nabila Khondakar, K T Nishant, Eric AlaniAbstract:MLH1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how MLH1-Mlh3 functions in both meiosis and MMR, we analyzed in bakers yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. MLH1-mlh3 representatives for each separation of function class were purified and characterized. Both MLH1-mlh3-32 (MMR+, crossover-) and MLH1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with MLH1-Mlh3 in MMR, stimulated the endonuclease activity of MLH1-mlh3-32 but not MLH1-mlh3-45, suggesting that MLH1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for two mlh3 mutants with opposite separation of function phenotypes, and an endonuclease defective mutant. Unexpectedly, all three showed increases in the number of non-crossover events that were not observed in mlh3null. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating MLH1-Mlh3 enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.
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MLH1-Mlh3 binding to mismatched and Holliday junction substrates inhibit its endonuclease activity.
2017Co-Authors: Carol M Manhart, Joaquin Ortega, Jennifer A Surtees, Martin A. White, Eric AlaniAbstract:In native agarose gels, migration of nicked product (n), linear product (black triangle), and closed circular substrate (cc) are indicated. All endonuclease reactions were carried out for 60 min, then stopped by addition of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and ProteinaseK as described in the Materials and methods. (A) Endonuclease activity was performed with supercoiled pUC18 as described in the Materials and methods. Where + Mg2+ or + Mn2+ is indicated, 1 mM MgCl2 or MnSO4 was added. Where both Mg2+ and Mn2+ are indicated, 0.5 mM of each was included. Where + MLH1-Mlh3 is indicated, 300 nM wild type or MLH1-mlh3D523N was added. (B) MLH1-Mlh3 endonuclease activity on a 2.7 kb circular DNA substrate is inhibited by preincubating MLH1-Mlh3 with oligonucleotide substrates. MLH1-Mlh3 (100 nM) was preincubated with increasing amounts of ~50 bp double-stranded oligonucleotide substrates for 15 min at 30°C (0–2,000 nM): either homoduplex (0 μM, 10 μM, 25 μM, 50 μM, 100 μM, or 200 μM, expressed as total nucleotide concentration), +8 loop (0 μM, 10 μM, 25 μM, 50 μM, 100 μM, or 200 μM, expressed as total nucleotide concentration), or 30 bp armed Holliday junction (0 μM, 24 μM, 60 μM, 120 μM, 240 μM, or 480 μM, expressed as total nucleotide concentration). After the preincubation step, reactions were challenged with ~18 μM (expressed as total nucleotide concentration) 2.7 kb circular substrate and incubated by conditions described for endonuclease assays in the Materials and methods and analyzed by agarose gel. All lanes contain 1 mM Mg2+. (C) Average of quantification of plasmid nicked for four separate experiments from B; error bars represent standard deviation.
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Site directed mutagenesis of MLH3.
2017Co-Authors: Najla Al-sweel, Carol M Manhart, Jennifer A Surtees, Vandana Raghavan, Abhishek Dutta, V P Ajith, Luigi Di Vietro, Nabila Khondakar, K T Nishant, Eric AlaniAbstract:A. Functional organization of Mlh3 based on sequence homology and secondary structure prediction [51]. The vertical bars indicate the approximate position of the mlh3 mutations (except mlh3-60) analyzed in this study and described in panel B. mlh3-39, -40, -57, -58, and -59 colored in red are based on highly conserved residues in the endonuclease motifs of Pms1 which were shown in the crystal structure of MLH1-Pms1 to form a single metal binding site [51] described in panel C. B. Amino acid positions of charged-to-alanine substitutions presented in red on the primary sequence of Saccharomyces cerevisiae Mlh3. Each cluster of underlined residues represents one allele corresponding to the vertical bars in panel A. mlh3-39, -40, -57, -58, and -59 are colored in red as in panel A. mlh3-60 represents the last 11 residues of Pms1 which constitute patch II of the heterodimerization interface of MLH1-Pms1 [51]. C. Metal binding site of Pms1 (left panel) from [51] comprised of the five highlighted residues (H703, E707, C817, C848, and H850) were found to be highly conserved in Mlh3 (right panel) based on sequence alignment and structural modeling (H525, E529, C670, C701, and H703) and were targeted in the mutagenesis described in this study (alleles represented in red in A and B).
Angela Brieger - One of the best experts on this subject based on the ideXlab platform.
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Loss of MLH1 sensitizes colon cancer cells to DNA-PKcs inhibitor KU60648.
Molecular carcinogenesis, 2017Co-Authors: Inga Hinrichsen, Stefan Zeuzem, Anne Ackermann, Tonja Düding, Annika Graband, Natalie Filmann, Guido Plotz, Angela BriegerAbstract:Germline mutations of MLH1 are responsible for tumor generation in nearly 50% of patients with Lynch Syndrome, and around 15% of sporadic colorectal cancers show MLH1-deficiency due to promotor hypermethylation. Although these tumors are of lower aggressiveness the benefit for these patients from standard chemotherapy is still under discussion. Recently, it was shown that the sensitivity to the DNA-PKcs inhibitor KU60648 is linked to loss of the MMR protein MSH3. However, loss of MSH3 is rather secondary, as a consequence of MMR-deficiency, and frequently detectable in MLH1-deficient tumors. Therefore, we examined the expression of MLH1, MSH2, MSH6, and MSH3 in different MMR-deficient and proficient cell lines and determined their sensitivity to KU60648 by analyzing cell viability and survival. MLH1-dependent ability of double strand break (DSB) repair was monitored after irradiation via γH2AX detection. A panel of 12 colon cancer cell lines, two pairs of cells, where MLH1 knock down was compared to controls with the same genetic background, and one MLH1-deficient cell line where MLH1 was overexpressed, were included. In summary, we found that MLH1 and/or MSH3-deficient cells exhibited a significantly higher sensitivity to KU60648 than MMR-proficient cells and that overexpression of MLH1 in MLH1-deficient cells resulted in a decrease of cell sensitivity. KU60648 efficiency seems to be associated with reduced DSB repair capacity. Since the molecular testing of colon tumors for MLH1 expression is a clinical standard we believe that MLH1 is a much better marker and a greater number of patients would benefit from KU60648 treatment.
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Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1
Molecular cancer, 2014Co-Authors: Inga Hinrichsen, S Passmann, Stefan Zeuzem, Nicolaus Friedrichs, Verena Steinke, Guido Plotz, Benjamin Philipp Ernst, Franziska Nuber, Dieter Schäfer, Angela BriegerAbstract:Introduction Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated.
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cytoskeletal scaffolding proteins interact with lynch syndrome associated mismatch repair protein MLH1
Proteomics, 2010Co-Authors: Angela Brieger, S Passmann, Fabian Wolpert, Boris Adryan, Stefan Zeuzem, Jorg TrojanAbstract:: The involvement of MLH1 in several mismatch repair-independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two-hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the "General Repository for Interaction Datasets" highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin beta 4 and Actin gamma, Cathepsin B and Annexin A2 as well as Spectrin alpha and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1-deficient and -proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1-Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.
