Journal: eLife

Article Title: Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

doi: 10.7554/eLife.21900

Figure Lengend Snippet: ( a ) Crystal structure of the C-terminal region of S. cerevisiae Mlh1-Pms1 heterodimer (pdb code 4FM0) . The Mlh1 and Pms1 regions are colored in green and yellow, respectively. The two metal ions of the endonuclease site are represented by grey spheres. The peptide containing the Mlh1-binding motif for Exo1 is colored in magenta. ( b ) Mutator assay. Frequency of reversion to Lys+ in cells containing the indicated MLH1 genotype at its endogenous locus. Values are the mean of 9 independent colonies ± SEM. ( c ) Spore viability of diploid SK1 strains bearing the indicated MLH1 genotype at its endogenous locus. MLH1 : VBD1311 (188 tetrads), Mlh1-HA: VBD1456 (22 tetrads), Mlh1-Flag: VBD1337 (26 tetrads), mlh1∆ : VBD1494 (178 tetrads). ***p<5.10 −5 , Fisher’s exact test. ( d ) Crossing over frequency at the HIS4LEU2 hotspot monitored by Southern blot at the indicated times in meiosis. Positions of parental bands (P1 and P2) and of the recombinant crossover products (CO1 and CO2) are indicated. Graph shows quantification at 9 hr from two independent biological replicates ± SEM. Same strains as in ( c ). DOI: http://dx.doi.org/10.7554/eLife.21900.003

Article Snippet: Full length mouse HFM1, MLH1, MLH3 cDNA were amplified from plasmids (obtained from Genscript, Genocopia and Genocopia, respectively) and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc’his.

Techniques: Binding Assay, Southern Blot, Recombinant

Journal: eLife

Article Title: Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

doi: 10.7554/eLife.21900

Figure Lengend Snippet: ( a ) Affinity pull-down of Mlh1-Flag from cells at 4 hr in meiosis. M: marker, No tag: VBD1311, Mlh1-Flag: VBD1337. Left: silver-stained gel of pulled-down proteins. Right: mass-spectrometry analysis of the most significant proteins pulled-down with Mlh1. One representative experiment is shown. ( b ) Reciprocal co-immunoprecipitation between Mer3-Flag and Mlh1-HA from meiotic cells at 4 hr in meiosis, analyzed by Western blot. Mer3-Flag: VBD1420, Mlh1-HA: VBD1456, Mer3-Flag Mlh1-HA: VBD1454. ( c ) Comparative analysis of two-hybrid interactions and co-IP in meiotic cells. The domain limits are based on Mer3 modeling with the Mer3 family-related Brr2 helicase structure . nd: not determined. ( d ) Co-IP between Mer3-Flag and Mlh1-HA at 4 hr in meiosis, in the presence or absence of MutLβ component Mlh2. Mlh1-HA: VBD1456, Mer3-Flag Mlh1-HA: VBD1454, Mer3∆C-Flag Mlh1-HA: VBD1490, Mer3-Flag Mlh1-HA mlh2∆ : VBD1550, Mer3∆C-Flag Mlh1-HA mlh2∆ : VBD1552. DOI: http://dx.doi.org/10.7554/eLife.21900.004

Article Snippet: Full length mouse HFM1, MLH1, MLH3 cDNA were amplified from plasmids (obtained from Genscript, Genocopia and Genocopia, respectively) and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc’his.

Techniques: Marker, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay

Journal: eLife

Article Title: Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

doi: 10.7554/eLife.21900

Figure Lengend Snippet: Serial dilutions of strains expressing the different fusion proteins were plated on minimal media lacking the indicated aminoacids to select for interactions. ( a ) Interaction between the S. cerevisiae proteins or their domains with the indicated coordinates. Mlh1 and Mlh3 interaction is used as a positive control. ( b ) Interaction between the mouse proteins. mMLH1 and mMLH3 interaction is used as a positive control. Below: diagram showing the tested mHFM1 domains for their interaction with mMLH1. Domains are color-coded as in . ( c ) Co-IP between Mer3-Flag and Mlh1-HA from meiotic cells at 4 hr in meiosis, analyzed by Western blot. Mlh1-HA: VBD1564, Mer3-Flag Mlh1-HA: VBD1576, Mer3∆IG-Flag Mlh1-HA: VBD1579. ( d ) Affinity pull-down of Mer3-Flag from cells at 4 hr in meiosis. No tag: VBD1311, Mer3-Flag: VBD1420. Top: silver-stained gel of pulled-down proteins. Bottom: mass-spectrometry analysis of the MutL proteins pulled-down with Mer3. One representative experiment is shown. DOI: http://dx.doi.org/10.7554/eLife.21900.005

Article Snippet: Full length mouse HFM1, MLH1, MLH3 cDNA were amplified from plasmids (obtained from Genscript, Genocopia and Genocopia, respectively) and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc’his.

Techniques: Expressing, Positive Control, Co-Immunoprecipitation Assay, Western Blot, Staining, Mass Spectrometry

Journal: eLife

Article Title: Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

doi: 10.7554/eLife.21900

Figure Lengend Snippet: ( a ) Silver-stained gel showing the direct interaction between purified Mer3 and Mlh1-Mlh2 (MutLβ) complex tagged with MBP on Mlh2. MBP-tagged MutLβ or MBP were bound to amylose resin and incubated with Mer3-His in the presence of 80 mM NaCl. Proteins were eluted with maltose. ( b ) Western Blot analysis using anti His antibody showing the pulldown of purified Mer3-His by GST-Mlh1 or by MBP-Mlh2 in the presence of 150 mM NaCl. Both panels are from the same exposure time of the same membrane. ( c ) Structural model of the IG-like domain of Mer3, based on Brr2 helicase structure . Top diagram: surface of the IG-like domain showing aminoacid conservation, from low (white) to high (red). Bottom: Electrostatic potential indicated by a color code, from positive (blue) to negative (red) charge. The position of Arg893 is indicated. ( d ) Western Blot showing the pulldown of purified Mer3-His or Mer3R893E-His by MBP-Mlh2 or MBP alone as a control. Pulldown was done in the presence of 150 mM NaCl. ( e ) Helicase assays with Mer3 or Mer3R893E on labeled D-loop substrate. 'Heat’, heat-denatured DNA substrate indicates the position of ssDNA. Assays were performed with or without RPA (20 nM) as indicated. DOI: http://dx.doi.org/10.7554/eLife.21900.006

Article Snippet: Full length mouse HFM1, MLH1, MLH3 cDNA were amplified from plasmids (obtained from Genscript, Genocopia and Genocopia, respectively) and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc’his.

Techniques: Staining, Purification, Incubation, Western Blot, Membrane, Control, Labeling

Journal: eLife

Article Title: Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

doi: 10.7554/eLife.21900

Figure Lengend Snippet: ( a ) Scheme of Mlh2 and Mlh1 expression constructs. MBP, Maltose binding protein tag; His, 8x histidine tag; PP site, PreScission Protease cleavage site. Right: 4% to 15% SDS-PAGE showing samples from a representative purification of Mlh1-Mlh2. The MBP tag was cleaved by using PreScission protease. We observed a single band for Mlh1, which migrated at a position corresponding to its molecular weight of 87 kDa. The 78 kDa-large Mlh2 polypeptide co-migrated with Mlh1, with a fraction of the protein (~20%) migrating above it. ( b ) Scheme of Mer3 expression construct. MBP, Maltose binding protein tag; His, 10x histidine tag. A 7.5% SDS-PAGE showing various fractions from Mer3 purification. The MBP tag was cleaved by using PreScission protease. ( c ) Silver-stained gel showing the direct interaction between purified Mer3 and Mlh2. MBP-Mlh2 or MBP alone was incubated with Mer3-His in the presence of 80 mM NaCl. MBP 1 is expressed and purified from E.coli and MBP 2 results from the cleavage of MBP tag from Mlh2 protein. ( d ) Silver-stained gel showing the direct interaction between purified Mer3 and Mlh1. GST-Mlh1 or GST resin was incubated with Mer3-His in the presence of 80 mM NaCl. DOI: http://dx.doi.org/10.7554/eLife.21900.007

Article Snippet: Full length mouse HFM1, MLH1, MLH3 cDNA were amplified from plasmids (obtained from Genscript, Genocopia and Genocopia, respectively) and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc’his.

Techniques: Expressing, Construct, Binding Assay, SDS Page, Purification, Molecular Weight, Staining, Incubation

Journal: eLife

Article Title: Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

doi: 10.7554/eLife.21900

Figure Lengend Snippet: ( a ) and ( b ): Serial dilutions of strains expressing the different fusion proteins were plated on minimal media lacking the indicated aminoacids to select for interactions. ( a ) Interaction between the S. cerevisiae proteins. Mlh1 and Mlh2 interaction is used as a positive control. ( b ) Interaction between the mouse proteins or their domains with the indicated coordinates. mMLH1 and mPMS1 interaction is used as a positive control. Below: diagram showing the tested mHFM1 domain for its interaction with mPMS1. Domains are color-coded as in . DOI: http://dx.doi.org/10.7554/eLife.21900.009

Article Snippet: Full length mouse HFM1, MLH1, MLH3 cDNA were amplified from plasmids (obtained from Genscript, Genocopia and Genocopia, respectively) and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc’his.

Techniques: Expressing, Positive Control

Journal: eLife

Article Title: Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

doi: 10.7554/eLife.21900

Figure Lengend Snippet: ( a ) Reciprocal co-IP between Mer3-Flag and Mlh2-Myc from meiotic cells at 4 hr in meiosis, analyzed by Western blot. Mer3-Flag: VBD1420, Mlh2-Myc: VBD1628, Mer3-Flag Mlh2-Myc: VBD1670. ( b ) Co-IP between Mer3- or Mer3R893E-Flag and Mlh2-Myc or Mlh1-HA from meiotic cells at 4 hr in meiosis, analyzed by Western blot. Mlh1-HA Mlh2-Myc: VBD1630; Mer3-Flag Mlh1-HA Mlh2-Myc: VBD1629; Mer3R893E-Flag Mlh1-HA Mlh2-Myc: VBD1681. ( c ) Mlh2-Myc levels at three meiotic recombination hotspots assessed by ChIP and qPCR at the indicated times during a meiotic time-course (VBD1670). ( d ) Same as in ( c ) but in a DSB-deficient spo11∆ strain (VBD1702). ( e ) Same as in ( c ) but in the mer3R893E strain (VBD1637). ( f ) Effect of polymorphism at the HIS4LEU2 hotspot on Mlh2 binding. Mlh2-Myc association with the HIS4LEU2 hotspot was assessed by ChIP at the indicated times in meiosis in strains containing either the wild-type or R893E mer3 allele, in strains with one base mismatch or without a mismatch at the HIS4LEU2 hotspot. WT one base mismatch: VBD1670; WT no mismatch: VBD1710; mer3R893E one base mismatch: VBD1637; mer3R893E no mismatch: VBD1706. ( c ), ( e ) and ( f ): Values are the mean ± SEM from two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.21900.011

Article Snippet: Full length mouse HFM1, MLH1, MLH3 cDNA were amplified from plasmids (obtained from Genscript, Genocopia and Genocopia, respectively) and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc’his.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Binding Assay

Journal: eLife

Article Title: Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

doi: 10.7554/eLife.21900

Figure Lengend Snippet: ( a ) Nuclease assay was performed with Mlh1-Mlh2 (as indicated) or Mlh1-Mlh3 (300 nM) on the indicated super-coiled circular plasmid DNA substrate (scDNA). Mlh1-Mlh3 is shown as a positive control producing nicked circular DNA ( b ) Quantitation of electrophoretic mobility shift assays with Mlh1-Mlh2 and various oligonucleotide-based DNA substrates in the presence of magnesium (2 mM). Values are the mean ± SEM from two independent experiments. ( c ) Electrophoretic mobility shift assays with Mer3 and the indicated DNA substrates, in the presence of either 2 mM magnesium (+Mg 2+ ) or 3 mM EDTA (–Mg 2+ ). Values are the mean ± SEM from two to tree independent experiments. DOI: http://dx.doi.org/10.7554/eLife.21900.013

Article Snippet: Full length mouse HFM1, MLH1, MLH3 cDNA were amplified from plasmids (obtained from Genscript, Genocopia and Genocopia, respectively) and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc’his.

Techniques: Nuclease Assay, Plasmid Preparation, Positive Control, Quantitation Assay, Electrophoretic Mobility Shift Assay

Journal: PLoS Biology

Article Title: A novel function for CDK2 activity at meiotic crossover sites

doi: 10.1371/journal.pbio.3000903

Figure Lengend Snippet: Chromosome spread preparations from adult (postnatal day 40) testes immunostained for MLH1 (green) and SYCP3 (red) are shown for WT (A–D) and Cdk2 T160A (E–F) for selected stages of meiotic prophase I. Positivity for MLH1 is seen in mid-pachytene through diplotene in WT (C, D) but could not be detected in any Cdk2 T160A stage. Images are representative of at least 20 images taken for equivalent stages. Identical staining patterns were confirmed in at least 3 biological replicates. Scale bars are representative of 5 μm. MLH1 foci were quantified specifically for mid-pachytene, late-pachytene, and diplotene/diplotene-like stages (G) by counting the average numbers of MLH1 foci per nucleus. Data are presented as individual foci counts for WT (orange bars, N = 90 for mid-pachytene, late-pachytene, and diplotene stages) and Cdk2 T160A (blue bars, N = 72 for mid-pachytene and N = 60 for diplotene-like stages; late-pachytene stages were not detected in this genotype). Error bars are indicative of the mean and SD. All data were assumed to be non-normally distributed. Statistical significance between genotypes was determined by unpaired t test. Significance and P -values are reported directly over each comparison. The underlying data for (G) can be found in . CDK2, cyclin-dependent kinase 2; MLH1, MutL homolog 1; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type.

Article Snippet: We measured interfocus distances of MLH1 foci and fitted data to a gamma distribution [ ].

Techniques: Staining, Comparison, Standard Deviation

Journal: PLoS Biology

Article Title: A novel function for CDK2 activity at meiotic crossover sites

doi: 10.1371/journal.pbio.3000903

Figure Lengend Snippet: Chromosome spread preparations from pubertal (postnatal day 25) testes immunostained for CDK2 or MLH1 (green) and SYCP3 (red) are shown for WT (A,D), Cdk2 Y15S (B,E), and Cdk2 Y15S/T160A (C,F) pachytene spermatocytes. Interstitial CDK2 and MLH1 foci can be detected in Cdk2 Y15S , but not Cdk2 Y15S/T160A , spermatocytes, which do not reach the equivalent meiotic stage. Images are representative of at least 20 nuclei imaged at equivalent stages. Identical staining patterns were confirmed in at least 3 biological replicates. Scale bars = 5 μm. Quantification of MLH1 foci, CDK2 foci, and frequency of homologs with 2 or 3 foci in indicated genotypes are shown in panels G, H, and I, respectively. Gamma distribution analysis of MLH1 foci along bivalents (J). Giemsa staining of diakinesis preparations from WT and Cdk2 Y15S/Y15S gonads showing comparable chiasmata with 20 bivalent chromosomes in either genotype (K). Chiasmata numbers for each cell as well as average counts (+SD) for WT and Y15S are shown in (L). Data from age-matched WT animals are shown for comparison. Individual points are pooled from at least 3 biological replicates; error bars are representative of SD. Interfocus distances between MLH1 foci on (chromosomes with >1 MLH1 focus) are quantified for each genotype in panel J for WT and Cdk2 Y15S spermatocytes. The underlying data for (G, H, I, J, L) can be found in . CDK2, cyclin-dependent kinase 2; MLH1, MutL homolog 1; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type; YS, CDK2 Y15S genotype; YS-TA, CDK2 Y15S/T160A genotype.

Article Snippet: We measured interfocus distances of MLH1 foci and fitted data to a gamma distribution [ ].

Techniques: Staining, Comparison, Standard Deviation

Journal: Regenerative Therapy

Article Title: Increased error-free DNA repair gene expression through reprogramming in human iPS cells ☆

doi: 10.1016/j.reth.2019.06.003

Figure Lengend Snippet: Comparative microarray and RT-qPCR analysis in the iPS cell lines and the parental HDF and 10YP cells: HDF clone1 (HDF-1), HDF clone2 (HDF-8), 10YP clone1 (10YP-15 cl1-39), and 10YP clone2 (10YP15 cl1-58). For PARP1, PARP2, PARG, RAD51, BLM, MSH2, MSH6, and MLH1, we include the RT-qPCR data (shown * line under the microarray data) along with the microarray data.

Article Snippet: MSH2 mouse monoclonal, (abcam, ab52266, 1:200 dilution), MSH6 mouse polyclonal, (BD Biosciences, 610919, 1:200 dilution), MLH1 (abnova, H00004292-M02, 1:200 dilution).

Techniques: Microarray, Activity Assay, Translesion Synthesis

Journal: Regenerative Therapy

Article Title: Increased error-free DNA repair gene expression through reprogramming in human iPS cells ☆

doi: 10.1016/j.reth.2019.06.003

Figure Lengend Snippet: The protein levels of RAD51 and BLM (HR); NBS1, MRE11, and RAD50 (HR); KU70 and KU80 (NHEJ); MSH2, MSH6, and MLH1 (MMR) were analyzed via Western blotting. β-actin was used as a loading control.

Article Snippet: MSH2 mouse monoclonal, (abcam, ab52266, 1:200 dilution), MSH6 mouse polyclonal, (BD Biosciences, 610919, 1:200 dilution), MLH1 (abnova, H00004292-M02, 1:200 dilution).

Techniques: Western Blot, Control