mutant synthetic 60-mer fan1 peptide  (GenScript corporation)

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) Top: Protein domain architecture of human FAN1 indicating the location of the MIP box. PIP, proliferating cell nuclear antigen–interacting peptide box; UBZ, ubiquitin-binding zinc finger; SAP, SAF-A/B, Acinus, and PIAS; TPR, tetratricopeptide repeat; NUC, nuclease. Bottom: Sequence alignment of the FAN1 orthologs with the MIP box of human EXO1 and BLM. ( B ) Dissociation constants ( K d ) indicating the binding affinity of MLH1 CTD for the indicated peptides from FAN1, EXO1, and BLM as determined by ITC. NI, no interaction (weak and constant signal under the condition used). ( C ) Immunoblots of FLAG-M2 affinity resin immunoprecipitations (IPs) of extracts from HEK293 cells transfected either with empty vector (e.v.), FLAG-FAN1 wild type (wt), or the variant constructs. The Y128A/F129A mutation is denoted as MIP*. ( D ) Immunoblots of FLAG-M2 affinity resin IPs of extracts from HEK293 cells transfected with the indicated FLAG-FAN1 constructs. ( E ) Immunoblots of FLAG-M2 affinity resin IPs of extracts from HEK293 cells cotransfected with FLAG-FAN1 and vectors expressing hemagglutinin (HA)–tagged dominant-negative (dn) forms of CDK1 or CDK2. ( F ) Immunoblots of green fluorescent protein (GFP)–Trap IPs of extracts from HeLa GFP-FAN1 cells synchronized in mitosis by thymidine-nocodazole block. Mitotic cells were replated in fresh medium for the indicated time periods. The relative ratios of pS126 to FAN1 and MLH1 to FAN1 in GFP-Trap samples were quantified by densitometry using ImageJ. (C to F) The antibodies used are shown on the left.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Ubiquitin Proteomics, Binding Assay, Sequencing, Western Blot, Transfection, Plasmid Preparation, Variant Assay, Construct, Mutagenesis, Expressing, Dominant Negative Mutation, Blocking Assay

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) Top: Protein domain architecture of human FAN1 indicating the location of the MIP box and MIM. Bottom: Sequence alignment of FAN1 orthologs with the putative MIM of human MLH3, PMS2, and PMS1. ( B ) ITC was used to determine the K d indicating the binding affinity of MLH1-CTD for the indicated peptides. ( C ) Immunoblots of FLAG-M2 affinity resin IPs of extracts from HEK293 cells transfected either with e.v. or indicated FLAG-FAN1 expression constructs. The L155A/L159A mutation is denoted as MIM*. ( D ) Immunoblots of FLAG-M2 affinity resin IPs of extracts from HEK293 cells transfected either with e.v. or indicated FLAG-FAN1 expression constructs. ( E and F ) Recombinant MutLα (E) or MutLγ (F) (200 nM) was subjected to pull-down reactions using the indicated recombinant GST-FAN1 (amino acids 118 to 177) variants. (C to F) The antibodies used are shown on the left.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Sequencing, Binding Assay, Western Blot, Transfection, Expressing, Construct, Mutagenesis, Recombinant

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) PLA was used to evaluate FAN1-MLH1 association in U2OS GFP-FAN1 wt and U2OS GFP-FAN1 MIP*/MIM* cells mock-treated or treated with MMC (150 ng/ml) for 24 hours. Representative images are shown. Scale bars, 10 μm. Scatterplot displays quantification of the PLA signals per nucleus from at least 100 cells. Data display the means ± SD from three independent experiments. Statistical significance was calculated by unpaired t test. ** P < 0.01; ns, not significant. ( B ) Schematic representation of human FAN1, highlighting positions of mutations used, including MIP*, MIM*, dimerization-defective (dim*), and nuclease-defective (nd*) FAN1 variants. ( C and D ) Immunoblots of GFP-Trap IPs of extracts from indicated U2OS GFP-FAN1 cells. The antibodies used are shown on the left. ( E ) Clonogenic survival assay of the indicated U2OS GFP-FAN1 cells exposed to increasing doses of MMC. Viability of untreated cells was defined as 100%. Data are presented as the means ± SEM.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Western Blot, Clonogenic Cell Survival Assay

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) QIBC analysis of GFP-FAN1 foci in U2OS GFP-FAN1 cells mock-treated, treated with MMC (20 ng/ml) for 24 hours, or MMC-treated and then released for 72 hours. Color-coded scatterplots indicate the number of GFP-FAN1 foci per nucleus. ut, untreated. ( B ) Same cells as in (A) were pulse-labeled with ethynyl deoxyuridine (EdU) during the last 30 min before harvesting and subjected to the Click-IT reaction. Cell cycle distribution was evaluated by QIBC using the 4′,6-diamidino-2-phenylindole (DAPI) and EdU signals (fig. S5C). ( C ) QIBC of RPA2 foci in U2OS GFP-FAN1 cells mock-treated, treated with MMC (20 ng/ml) for 24 hours, or MMC-treated and then released for 72 hours. Color-coded scatterplots indicate the number of RPA2 foci per nucleus. A.U., arbitrary units. ( D ) Same cells as in (C) were treated with MMC (300 ng/ml) for 24 hours, and lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk indicates hyperphosphorylated form of RPA2.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Labeling, Western Blot

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) Scheme of the slipped (CTG)30/(CAG)50 substrate. ( B ) The (CAG)20 slip-out substrate was incubated with extracts from U2OS GFP-FAN1 cells expressing indicated FAN1 variants. Repair efficiency was quantified by densitometric analysis of Southern blots. Values represent the mean of three independent experiments. Error bars represent ±SD. Statistical significance was calculated by ordinary one-way analysis of variance (ANOVA) test followed by Tukey’s multiple comparisons. ** P < 0.005, *** P < 0.0005, **** P < 0.00005. ( C ) HeLa nuclear extract was incubated with FAN1-derived 60-mer peptides (amino acids 118 to 177) containing wt or mutant MIP-MIM, immunoprecipitated using anti-FAN1 antibody, and immunoblotted with the indicated antibodies. ( D ) Extracts from HeLa cells were supplemented with increasing concentrations of FAN1 peptides and incubated with the (CAG)20 slip-out substrate. Repair efficiency was calculated as described in (B). ( E ) Extracts of U2OS GFP-FAN1 wt and U2OS GFP-FAN1 MIP*/MIM* cells were supplemented with indicated FAN1 peptides and incubated with the (CAG)20 slip-out substrate. Repair efficiency was calculated as described in (B).

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Incubation, Expressing, Derivative Assay, Mutagenesis, Immunoprecipitation

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: Summary of the FAN1-MLH1 complex interaction and a plausible model for its involvement in ICL and slip-out repair. ( A ) Scheme of the FAN1-MLH1 interaction in different states of wt or mutated FAN1. Protein orientation is unknown and arbitrarily presented for ease. ( B ) Left: FAN1 is subjected to CDK-mediated phosphorylation at S126 located within the MIP box. Right: Regulation of FAN1-MLH1 interaction through the cell cycle and in response to ICL damage. ( C ) Top: The FAN1-MLH1 complex localizes to ICL damaged chromatin to preserve genome stability and ensure cell viability. In cells, devoid of the FAN1-MLH1 interaction aberrant ICL repair and cell death ensues. Inactivation of FAN1’s endo- and exonuclease activities by mutating D960A or inhibition of FAN1 dimerization, affecting FAN1’s endo- but not exonucleolytic activity, would alleviate the toxicity of the FAN1 MIP/MIM-mutated variant and promote cell proliferation and survival. Bottom: Inhibition of the FAN1-MLH1 interaction (MIP*/MIM*) blocks repair of slipped-DNAs. Repair defects were rescued when the MLH1-interacting–defective FAN1 was also defective either in both its endonuclease activities or in dimer formation (endo- but not exonuclease defective). Therefore, the regulatory aspects of the FAN1 binding to MLH1 aligns the pathway of ICL repair with that of slipped-DNA processing. Mutations: MIP*, Y128A/F129A; MIM*, L155A/L159A; nd*, D960A; dim*, K525E/R526E/K528E.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Phospho-proteomics, Inhibition, Activity Assay, Variant Assay, Binding Assay