Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Cross-analysis of the methylation of RAD51 and BRCA1 genes in tumor and non-tumor mucosa, considering the 37 cases with paired tumoral and non-tumoral samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Methylation

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in gastric adenocarcinoma and non-tumor gastric mucosa samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison of RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in tumor and non-tumor mucosa, considering the paired samples of the same individuals. The results are presented in box plots, using a logarithmic scale (+1 was added as a constant to allow the logarithmic representation of null values).

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in patients with previous neoadjuvant chemotherapy and in those without neoadjuvant chemotherapy, considering tumor samples and non-tumor mucosa samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Association between immunohistochemistry and mRNA levels in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Immunohistochemistry

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Correlation between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression and histological type, histological grade, perineural invasion, and vascular invasion in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Correlation between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression and depth of invasion (pT), regional lymph node metastasis, distant metastasis, and stage in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison between the mRNA levels in the tumors of patients who died and those who did not, considering the following groups: total population, excluding neoadjuvant therapy, with only surgical treatment, and with adjuvant or neoadjuvant treatment.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Adjuvant

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Kaplan-Meier curves comparing the overall survival of the 24 patients with tumor mRNA expression above the median with that of the 24 below the median, for RAD51 , ATR , BRCA1 , BRCA2 , and ATM .

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: PLoS Genetics

Article Title: Elevated temperature increases meiotic crossover frequency via the interfering (Type I) pathway in Arabidopsis thaliana

doi: 10.1371/journal.pgen.1007384

Figure Lengend Snippet: (A, B) γH2Ax foci counting. (A) Pollen mother cells containing chromosomes (DAPI, blue) and γH2Ax foci (red). (B) Boxplots of γH2Ax foci at 20˚C and 28˚C; mean shown as white circle. (C, D) RAD51 foci counting. (C) Pollen mother cells containing chromosomes (DAPI, blue) and RAD51 foci (red). (D) Boxplots of RAD51 foci at 20°C and 28°C; mean shown as white circle. p-values are indicated as follows: ns = p > 0.5, * = p ≤ 0.05, ** = p ≤ 0.01. Scale bars represent 5 μm.

Article Snippet: The slides were incubated overnight with primary antibodies diluted 1:200 (RAD51, γH2AX, MLH1) in blocking buffer (goat serum, AR0009, Bosterbio) at 4°C and then at 37°C for 60 min with secondary antibody (1:1000, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555, catalog # A-21428, Thermo Fisher Scientific).

Techniques:

Journal: Biomedicines

Article Title: USP7 Deregulation Impairs S Phase Specific DNA Repair after Irradiation in Breast Cancer Cells

doi: 10.3390/biomedicines12040762

Figure Lengend Snippet: USP7 inhibition and overexpression leads to increased DNA replication stress. ( A ): Examples of RAD51 foci or ( C ): RPA foci or ( E ): PCNA foci after 4/6 h with or without irradiation. ( B ): Quantification of RAD51 foci in RAD51 positive cells, 6 h or ( D ): of RPA-foci 4 h or ( F ): of PCNA foci 4 h after irradiation. Cells were treated with 4 µM P5091 and irradiated with 6 Gy. Fixation and immunofluorescence staining were performed 4/6 h after irradiation with the indicated antibodies plus nuclear staining using DAPI. Foci were quantified automatically by the Medipan AKLIDES ® device. ( G ): Exemplary depiction of EdU positive cells after irradiation, with or without USP7 inhibition. ( H ): EdU incorporation after irradiation. Cells were pulse-labeled with EdU and treated with either- or both P5091 and 6 Gy of ionizing radiation. Incorporated EdU was stained and EdU positive cells were captured. Fluorescence intensity was analyzed with the Image J software. Means are depicted in red. Shown are the means of three independent experiments ± SEM. Asterisks represent significant differences (n.s. not significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001, Student’s t -test).

Article Snippet: Foci were detected using the following primary antibodies: γH2AX (Ser139, Merck, Darmstadt, Germany #05-636, 1:250), 53BP1 (Novus, Centennial, CO, USA #NB100-305, 1:500), Rad51 (Merck #PC130, 1:500), PCNA (Cell Signaling #13110, 1:500) and RPA (Santa Cruz #sc53496).

Techniques: Inhibition, Over Expression, Irradiation, Immunofluorescence, Staining, Labeling, Fluorescence, Software

Journal:

Article Title: Coprinus cinereus rad50 Mutants Reveal an Essential Structural Role for Rad50 in Axial Element and Synaptonemal Complex Formation, Homolog Pairing and Meiotic Recombination

doi: 10.1534/genetics.108.092775

Figure Lengend Snippet: Anti-Rad51 staining in wild type and spo11-1. (A–E) Representative images of wild-type meiotic time course. (A) Preleptotene image taken 1 hr after karyogamy (K + 1). (B) Leptotene image taken at K + 2. (C) Zygotene image taken at K + 4. (D) Pachytene image taken at K + 6. (E) Diplotene image taken at K + 8. Bars, 2 μm for all images. (F) Mean number of Rad51 foci in spo11-1 (♦) and in wild type (□). Error bars show 95% confidence intervals.

Article Snippet: Purified Rad51 in SDS–PAGE gel slices was sent to Cocalico Biologicals (Reamstown, PA) and used for immunization of rabbits according to their injection schedule, using the acrylamide as the adjuvant.

Techniques: Staining

Journal:

Article Title: Coprinus cinereus rad50 Mutants Reveal an Essential Structural Role for Rad50 in Axial Element and Synaptonemal Complex Formation, Homolog Pairing and Meiotic Recombination

doi: 10.1534/genetics.108.092775

Figure Lengend Snippet: Mutants fall into two classes for Rad51 focus formation. (A) Mean number of Rad51 foci observed at K + 4. (B) Time course of Rad51 foci in rad50-14 and rad50-15. Wild-type data (□) are the same as in Figure 4 and shown for reference. •, rad50-14; ▴, rad50-15. Error bars show 95% confidence intervals.

Article Snippet: Purified Rad51 in SDS–PAGE gel slices was sent to Cocalico Biologicals (Reamstown, PA) and used for immunization of rabbits according to their injection schedule, using the acrylamide as the adjuvant.

Techniques:

Journal: bioRxiv

Article Title: Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

doi: 10.1101/2024.12.05.627070

Figure Lengend Snippet: A: General organization of Rad52 domains. The C-terminal domain is shown in black, with the region interacting with Rad51 in magenta, and the RPA binding region in orange B: Predicted disorder of Rad52-Cter domain (206-471) (see Materials and Methods). C: H- N SOFAST-HMQC spectrum of the uniformly N labelled Rad52-Cter domain (206-471) alone in grey and in blue, after addition of equimolar amount of unlabeled Rad51. D: Cα chemical shift index calculated for all assigned residues of the Rad52-Cter domain (206-471). The values close to zero of this index show that this domain is fully disordered, consistent with the predictions. E: Mapping of the interaction between Rad52-Cter domain (206-471) and Rad51, using the intensities ratio (I/I0), where I and I0 are the intensity of the signals 1H- N SOFAST-HMQC spectra before and after addition of Rad51, respectively. F: Sequence Logo of the central region of the Rad52-Cter domain (310-395) generated with homologues of Rad52 in fungi or in Saccharomycetales . F316 (Anchor 1), F337 (Anchor 2), and Y376 (Anchor 3) located in each conserved motif and mutated in this study are highlighted with a star.

Article Snippet: Whole-cell extracts (1 mg) were incubated (4°C for 1 hour) with 0.5 µl of anti-Rad51 polyclonal antibody (produced in rabbit by Eurogentec) for Rad51 immunoprecipitation.

Techniques: Binding Assay, Sequencing, Generated

Journal: bioRxiv

Article Title: Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

doi: 10.1101/2024.12.05.627070

Figure Lengend Snippet: A: Left panel: SEC profile analysis of Rad51, Rad52 (295-394) and Rad51+Rad52 (295-394) in a 1:1 ratio. Right panel: SDS-PAGE analysis of fraction from SEC profile in left panel revealed with coomassie blue B: SAXS data for the 1:1 complex between Rad51+Rad52 (295-408). Left panel: Series intensity (magenta, left axis) vs. elution volume, and, if available, radius of gyration (Rg) vs. elution volume (blue, right axis). The profile includes a major and a minor peak. The deconvolution of these peaks is shown in Supplementary Figure 1B. Right panel: Molecular weight calculated with SAXS data (Exp. MW) compared to theoretical MW (Calc. MW) for the major and minor peaks.

Article Snippet: Whole-cell extracts (1 mg) were incubated (4°C for 1 hour) with 0.5 µl of anti-Rad51 polyclonal antibody (produced in rabbit by Eurogentec) for Rad51 immunoprecipitation.

Techniques: SDS Page, Molecular Weight

Journal: bioRxiv

Article Title: Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

doi: 10.1101/2024.12.05.627070

Figure Lengend Snippet: A: Left panel: Best AlphaFold2 model of the complex between Rad51 (77–400) (grey surface) and Rad52 (310–394) (colored according to sequence conservation: from red for the highest conservation to white). Three boxes highlight zoomed-in regions shown in the right panels. Right panels: the side chains of Rad52 residues interacting with Rad51 are depicted as sticks, with mutated residues represented in ball-and-stick. Labels point to the C-alpha carbons of the mutated residues. B: AlphaFold2 model of the Rad51(77–400) complex with its surface colored according to sequence conservation and Rad52 (310–394) shown as a grey cartoon. Two perpendicular orientations are provided in the upper and lower panels, respectively. C: Interaction loss (%) plotted as a function of the surface area of each residue buried upon interaction. Interaction loss was determined by the intensity ratio between mutant and WT Rad52 co-immunoprecipitated with Rad51. D: Serial 10-fold dilutions of haploid strains bearing different RAD52 mutations of residue involved in the interaction, spotted onto rich medium (YPD) containing different MMS concentrations. Upper panel: SRS2 background, lower panel: srs2 Δ background.

Article Snippet: Whole-cell extracts (1 mg) were incubated (4°C for 1 hour) with 0.5 µl of anti-Rad51 polyclonal antibody (produced in rabbit by Eurogentec) for Rad51 immunoprecipitation.

Techniques: Sequencing, Residue, Mutagenesis, Immunoprecipitation

Journal: bioRxiv

Article Title: Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

doi: 10.1101/2024.12.05.627070

Figure Lengend Snippet: A: Left panel, best AlphaFold2 model of the complex between Rad51 (77–400) (grey surface) and Rad52 (310–394) (colored in magenta). The three key residues mutated are highlighted with balls. Right panel, structure of three Rad51 monomers assembled in a multimer (PDB code 1SZP). The central protein is truncated (Rad51 (70-400) and shown as a grey surface), the two other Rad51 molecules (#-1 et #+1) are shown as cartoon in blue. Residue F144 side chain is highlighted. For both panels, the Rad51 surface buried upon formation of the Rad51 filament is colored in blue. B: Zoomed-in of residues F316 and F337 (left panel) and Y376 (right panel). The Rad51 surface is shown with transparency to show the position of K214 and T161 from Rad51 contacting F337 and Y376, respectively. The Rad51 surface buried upon formation of the Rad51 filament is colored in blue. C: In light grey, Mapping of the interaction between Rad52-Cter domain mutants (295-394) and Rad51, using the intensities ratio (I/I0), where I and I0 are the intensity of the signals H- N SOFAST-HMQC spectra before and after addition of Rad51, respectively. For comparison, values for the WT are indicated in dark grey. D: SEC profile analysis of Rad51, Rad52 (295-394) and Rad51 + Rad52 (295-394) WT and mutants in a 1:1 ratio.

Article Snippet: Whole-cell extracts (1 mg) were incubated (4°C for 1 hour) with 0.5 µl of anti-Rad51 polyclonal antibody (produced in rabbit by Eurogentec) for Rad51 immunoprecipitation.

Techniques: Residue, Comparison

Journal: bioRxiv

Article Title: Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

doi: 10.1101/2024.12.05.627070

Figure Lengend Snippet: A: Survival curves of haploid cells with the indicated genotypes exposed to γ-rays. Survival from 1 to 100% is labeled in red to underline the difference of scale displayed on the left and the right panels. B: Survival rates of cells suffering an HO-induced gene conversion between MAT ectopic alleles. C: ChIP analysis shows that rad52-F316A (Anchor 1) and rad52-F337A (Anchor 2) highly impact Rad51 recruitment at a HO-induced DSB and the low dependency on Srs2. Upper panel: schematic of the HO-induced SSA repair system used. Lower panel: Rad51 ChIP relative enrichment was assessed at 0.6 kb from the DSB site 4 hours after HO induction. Data are presented as the median and minimum and maximum values of at least three independent experiments. Statistical analysis shows that deleting SRS2 in the rad52 mutants increase slightly but significantly the recruitment of Rad51 (* p < 0.05, two-tailed unpaired t-test). Already published data obtained with rad52-Y376A (Anchor 3) are shown for reference.

Article Snippet: Whole-cell extracts (1 mg) were incubated (4°C for 1 hour) with 0.5 µl of anti-Rad51 polyclonal antibody (produced in rabbit by Eurogentec) for Rad51 immunoprecipitation.

Techniques: Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

doi: 10.1101/2024.12.05.627070

Figure Lengend Snippet: A: Representative images of Rad51-iGFP 4hours after Isce-I induction in WT, srs2Δ , rad52 single mutants or rad52 srs2Δ double mutants. Maximum Z-projection is applied on fluorescent images. Minimum and maximum fluorescence intensities are indicated. Scale bar 1µm. Pie charts show the proportion of cells with different Rad51 structures as indicated. Bright foci correspond to mean foci intensities > 80% of WT, intermediate to mean values between 20 and 80% of WT, and dim to mean values < 20% of WT. Data are from at least 3 independent experimental replicates. The number of cells analyzed (n) is indicated below each pie chart. B: Comparison of Rad51 foci intensities, filament intensities and lengths in WT and mutant strains as indicated. The number of structures analyzed is indicated below each box. Intensities are normalized to the mean intensity of WT Rad51 filaments. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extent corresponds to the adjacent value, which is the most extreme data value not considered an outlier (above 75th percentile + 1.5 times interquartile range or below 25th percentile - 1.5 times interquartile range). Statistical test: logistic regression with binomial distribution. (See materials and methods; ***P < 0.001, **P < 0.01, *P < 0.05).

Article Snippet: Whole-cell extracts (1 mg) were incubated (4°C for 1 hour) with 0.5 µl of anti-Rad51 polyclonal antibody (produced in rabbit by Eurogentec) for Rad51 immunoprecipitation.

Techniques: Fluorescence, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

doi: 10.1101/2024.12.05.627070

Figure Lengend Snippet: A: Co-IP experiments showing the loss of interaction between Rad51-T161R and Rad52. Rad51 was immunoprecipitated with a poly-clonal anti-Rad51 antibody (αRad51). The presence of Rad51 in the immunoprecipitated fraction (IP) cannot be detected because it migrates at the same level as the anti-Rad51 IgG used for the immunoprecipitation. However, the absence of Rad52-FLAG in the rad51 Δ immunoprecipitate confirmed that the Rad52-FLAG signal observed is related to the Rad52–Rad51 interaction. Western blot analysis of Rad52-FLAG, Rad51 and Tubulin in whole cell extracts (WCE) reveals that the amount of proteins is comparable in each mutant strains. * Unspecific bands. B: Serial 10-fold dilutions of haploid strains with the indicated genotypes were spotted onto rich medium (YPD) containing different MMS concentrations. C: Survival curves of haploid cells with the indicated genotypes exposed to γ-Rays. D: ChIP was used to assess Rad51 relative enrichment at 0.6 kb from an HO cut site 4 hours after HO induction. Data are presented as the median and minimum and maximum values of at least three independent experiments. E: Left panel: Representative images of Rad51-iGFP 4hrs after Isce-I induction in WT, srs2 Δ, rad51-T161R or srs2 Δ rad51-T161R double mutants. Maximum Z-projection is applied on fluorescent images. Scale bar 1µm. Pie charts show the proportion of cells with different Rad51 structures as indicated. Bright foci correspond to mean foci intensities > 80% of WT, and Dim foci to mean values < 20% of WT. Data are from 3 independent experimental replicates. The number of cells analyzed (n) is indicated below each pie chart. Lower panel: Comparison of Rad51 filament intensities and lengths in WT and mutant strains as indicated. The number of structures analyzed is indicated below each box. Intensities are normalized to the mean intensity of WT Rad51 filaments. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extent corresponds to the adjacent value, which is the most extreme data value not considered an outlier (above 75th percentile + 1.5 times interquartile range or below 25th percentile - 1.5 times interquartile range). Statistical test, logistic regression with binomial distribution. (See materials and methods; ***P < 0.001, **P < 0.01, *P < 0.05).

Article Snippet: Whole-cell extracts (1 mg) were incubated (4°C for 1 hour) with 0.5 µl of anti-Rad51 polyclonal antibody (produced in rabbit by Eurogentec) for Rad51 immunoprecipitation.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

doi: 10.1101/2024.12.05.627070

Figure Lengend Snippet: Model depicting Rad52 assembly chaperone activity and protection against Srs2 through the interaction of anchors 1, 2 and 3 with Rad51.

Article Snippet: Whole-cell extracts (1 mg) were incubated (4°C for 1 hour) with 0.5 µl of anti-Rad51 polyclonal antibody (produced in rabbit by Eurogentec) for Rad51 immunoprecipitation.

Techniques: Activity Assay

Journal: PLOS Genetics

Article Title: Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

doi: 10.1371/journal.pgen.1010407

Figure Lengend Snippet: (A) Meiotic progression. WT (NH716 or NH2598 RCEN) ( n = 18) and hedΔR (NH2528 or NH2616 RCEN) ( n = 21) diploids were transferred to Spo medium. At the indicated timepoints cells were fixed, stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by fluorescent microscopy for the presence of binucleate (MI) or tetranucleate (MII) cells. The average values from different timecourses were plotted for each time point. At least 200 cells were counted for each timepoint. (B) Sporulation. Cells were examined by light microscopy for the presence of asci containing one to four spores. For each biological replicate, 200 cells were counted. (C) Quantification of meiotic progression timing. T 50 represents the amount of time (hr) it took for a sporulation culture from Panel A to reach one-half its maximum %MI+MII value. (D) Cytological assay for the presence of DSBs. Representative images of chromosome spreads generated from WT (NH716) and spo11Δ (NH1055) cells taken after four hours in Spo medium. DNA was visualized with DAPI and antibodies against Rad51 and Dmc1 were used to detect the presence of DSBs. Scale bar is 2 μm. (E) Quantification of percent cells exhibiting at least three Dmc1 and Rad51 foci. Chromosome spreads of WT ( n = 3), and hedΔR ( n = 3) diploids from a subset of the replicates shown in Panel A were analyzed for the presence of Rad51 and Dmc1 foci at various timepoints. Nuclei containing a minimum of three Rad51 and three Dmc1 foci were scored as positive. At least 120 nuclei were examined for each timepoint. (F) Lifespan of cells exhibiting both Rad51 and Dmc1 foci. Each time course from Panel E was graphed and the lifespan of Rad51/Dmc1 positive nuclei (hr) was calculated by dividing the area under each curve by the maximum number of cells that completed either MI or MII for the corresponding culture . (G) Examples of Red1 whole cell immunofluorescence in a WT meiotic time course. Scale bar is 2 μm. (H) Prophase I progression. Whole fixed cells from a subset of the replicates shown in Panel A (WT and hedΔR , n = 11) were assayed for the presence of Red1 by immunofluorescence and the average values were plotted. At least 200 cells were counted for each timepoint. (I) Quantification of prophase I length. Prophase I lifespan for each sporulation culture included in Panel H was calculated as described for Panel F. For all bar graphs, each dot represents a biological replicate, the height of the bar indicates the average, and the error bars indicate standard deviation. For all line graphs, error bars indicate standard deviation. Data were analyzed for statistical significance using an unpaired, two-tailed Student T-test. (* = p < .05; ** = p < .01; *** = p < .001)

Article Snippet: The Rad51 antibody was generated by Covance Research Products (now Labcorp) in guinea pigs using the peptide QKDGNDFKFNYQGDEC.

Techniques: Staining, Microscopy, Light Microscopy, Generated, Immunofluorescence, Standard Deviation, Two Tailed Test

Journal: PLOS Genetics

Article Title: Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

doi: 10.1371/journal.pgen.1010407

Figure Lengend Snippet: (A) Spore viability. WT (NH716 or NH2598 RCEN), hedΔR (NH2528 or NH2616 RCEN), hedΔR rad51-II3A (NH2566) and rad51-II3A (NH2618 or NH2666 RCEN) tetrads from both liquid and solid sporulation conditions were dissected to determine the percent of viable spores. For each replicate, at least 18 tetrads were dissected. (B) Meiotic progression. Timecourses of the WT, hedΔR , hedΔR rad51-II3A ( n = 10) and rad51-II3A ( n = 10) were analyzed for meiotic progression as described in . The average of all replicates is plotted. (C) Comparison of T 50 values of the individual timecourses shown in Panel B. (D) Prophase I progression. A subset of timecourses in panel A (WT and hedΔR , n = 11, rad51-II3A and hedΔR rad51-II3A , n = 8) were analyzed for prophase I progression as described in . The average of all replicates is plotted. (E) Prophase I lifespan for each sporulation culture in Panel D was calculated as described in . The data for WT and hedΔR used for Panels B and D are the same as shown in . The statistical significance of differences between strains in Panel A was determined using the Mann-Whitney test, while Panels C and E used an unpaired, two-tailed Student’s T-test. (* = p < .05; ** = p < .01; *** = p < .001, **** = p < .0001).

Article Snippet: The Rad51 antibody was generated by Covance Research Products (now Labcorp) in guinea pigs using the peptide QKDGNDFKFNYQGDEC.

Techniques: Comparison, MANN-WHITNEY, Two Tailed Test

Journal: PLOS Genetics

Article Title: Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

doi: 10.1371/journal.pgen.1010407

Figure Lengend Snippet: (A) Meiotic progression. Timecourses of the following diploids were analyzed for meiotic progression as described in : WT, hedΔR , NDT80-mid (NH2495 or NH2634 RCEN) ( n = 13) and hedΔR NDT80-mid (NH2505 or NH2610 RCEN) ( n = 25). The average of all replicates is plotted. (B) Comparison of T 50 values of timecourses shown in Panel A. (C) Prophase I progression. A subset of timecourses in panel A ( NDT80-mid , n = 11, hedΔR NDT80-mid , n = 15) were analyzed for prophase I progression as described in . WT and hedΔR data are from . The average of all replicates is plotted. (D) Prophase I lifespan for each sporulation culture in Panel C was calculated as described in . (E) Spore viability. Sporulated cells from both liquid and solid sporulation media were dissected to determine the percent of viable spores. At least 20 tetrads were dissected for each replicate. In addition to the diploids listed for Panel A, hedΔR NDT80-mid rad51-II3A (NH2705 RCEN) and rad51-II3A were included. (F) The distribution of viable spores among tetrads dissected in Panel E. (G) Schematic of the chromosome VIII homologs used to monitor nondisjunction. The tetrad on the left is an example of normal chromosome VIII segregation while MI nondisjunction (NDJ) occurred in the tetrad on the right. B = blue, R = red, Bl = black. (H) Frequency of chromosome VIII MI nondisjunction. Numbers above each bar indicate the total number of tetrads assayed. Panels A-E for WT, hedΔR , and rad51-II3A used the same data shown in Figs and . The statistical significance of differences between strains in Panels B and D was determined using an unpaired, two-tailed Student’s T-test, while the Mann-Whitney test was used for Panels E and H. (* = p < .05; ** = p < .01; *** = p < .001, **** = p < .0001)

Article Snippet: The Rad51 antibody was generated by Covance Research Products (now Labcorp) in guinea pigs using the peptide QKDGNDFKFNYQGDEC.

Techniques: Comparison, Two Tailed Test, MANN-WHITNEY

Journal: PLOS Genetics

Article Title: Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

doi: 10.1371/journal.pgen.1010407

Figure Lengend Snippet: (A) Physical analysis of recombination. Schematic of the HIS4LEU2 hotspot. The dotted line indicates the position of the DSB which is flanked by XhoI restriction sites. The NgoMIV site near the DSB site is also shown. (B) Cultures generated from independent single colonies of WT (NH716 and NS2598 RCEN), hedΔR (NH2528 and NH2H2616 RCEN), NDT80-mid (NH2634 RCEN), hedΔR NDT80-mid (NH2505 and NH2610), hedΔR NDT80-mid rad51-II3A (NH2705 RCEN) and rad51-II3A (NH2666 RCEN) were incubated for 10 hours in Spo medium. DNA was isolated, digested with XhoI and NgoMIV and crossovers and noncrossovers were analyzed by Southern blot as described in . A representative gel of an individual culture from each strain is shown. P1 and P2 represent the parental homologs. NCO1 and CO2 represent unambiguous non-crossover and crossover bands. The numbers for each lane are colored coded with the indicated strains in the legend. (C) Quantification of CO2 after 10 hours in Spo medium. (D) Quantification of NCO1 after 10 hours in Spo medium. The statistical significance of differences between strains in Panels C and D was determined using an unpaired, two-tailed Student’s T-test. (* = p < .05; ** = p < .01; *** = p < .001, **** = p < .0001). (E) Genetic analysis of crossover formation. Single colonies from WT (NH2598 RGC) ( n = 7; tetrads = 3374), hedΔR (NH2616 RGC) ( n = 7; tetrads = 3812), NDT80-mid (NH2634 RGC) ( n = 7; tetrads = 3592), hedΔR NDT80-mid (NH2610 RGC) ( n = 8; tetrads = 3899), pif1-md (NH2695 RGC)( n = 7; tetrads scored = 2974), hedΔR pif1-md (NH2702 RGC)( n = 6; tetrads = 2799) and hedΔR NDT80-mid pif1-md (NH2687 RGC)( n = 8; tetrads = 2605) were sporulated on solid medium and analyzed by fluorescence microscopy for crossovers in the CEN8-ARG4 and ARG4-THR1 intervals . Error bars show standard error. Since MI nondisjunction and nonparental ditype tetrads are indistinguishable in the CEN8-ARG4 interval , the frequency of nonparental ditypes was corrected using the MI nondisjunction frequency for hedΔR and NDT80-mid . No correction was necessary for WT because MI nondisjunction was not observed. All CEN8-ARG4 NPDs were assumed to be due to MI nondisjunction in strains with Chromosome VIII MI nondisjunction frequencies ≥ 9% . Map distances (cM) and standard errors were calculated using the Stahl lab online tools ( https://elizabethhousworth.com/StahlLabOnlineTools/ ). (F) Crossover interference ratios between the CEN8-ARG4 and ARG4-THR1 intervals were determined using the method described by Malkova, Swanson . Arrows above and below the chromosome VIII diagram indicate ARG4-THR1 and CEN8-ARG4 as the reference interval, respectively. Test interval/Reference interval map distance ratios significantly less than one are indicative of interference and p values are indicated by asterisks (*<0.05; ****<0.0001) (G-test).

Article Snippet: The Rad51 antibody was generated by Covance Research Products (now Labcorp) in guinea pigs using the peptide QKDGNDFKFNYQGDEC.

Techniques: Generated, Incubation, Isolation, Southern Blot, Two Tailed Test, Fluorescence, Microscopy

Journal: PLOS Genetics

Article Title: Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

doi: 10.1371/journal.pgen.1010407

Figure Lengend Snippet: (A) Spore viability was plotted versus prophase I length for WT ( n = 12), hedΔR ( n = 11), hedΔR rad51-II3A ( n = 8), NDT80-mid ( n = 11), hedΔR NDT80-mid ( n = 16) and hedΔR NDT80-mid rad51-II3A ( n = 11). (B) Chromosome VIII MI nondisjunction was plotted versus prophase I length for hedΔR NDT80-mid and hedΔR NDT80-mid rad51-II3A , using the same replicates as in Panel A. These data are from the same timecourses shown in Figs and . The correlation coefficient and statistical significance are indicated by “ r ” and “ p ”, respectively and were determined using GraphPad Prism 9.0.

Article Snippet: The Rad51 antibody was generated by Covance Research Products (now Labcorp) in guinea pigs using the peptide QKDGNDFKFNYQGDEC.

Techniques:

Journal: PLOS Genetics

Article Title: Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

doi: 10.1371/journal.pgen.1010407

Figure Lengend Snippet: (A) Spore viability. The data from WT, hedΔR , NDT80-mid and hedΔR NDT80-mid were taken from . Additional strains were pif1-md (NH2657 or NH2685 RCEN), rrm3Δ (NH2485::pRS304 2 or NH2623 RCEN), pif1-md rrm3Δ (NH2671::pRS304 2 ), hedΔR pif1-md (NH2691 or NH2702 RCEN), hedΔR rrm3Δ (NH2540 or NH2629 RCEN), hedΔR pif1-md rrm3Δ (NH2704), NDT80-mid pif1-md (NH2725), NDT80-mid rrm3Δ (NH2549), hedΔR NDT80-mid pif1-md (NH2661 or NH2687 RCEN), hedΔR NDT80-mid rrm3Δ (NH2596), and hedΔR NDT80-mid pif1-md rrm3Δ (NH2670). Sporulated cells from both liquid and solid sporulation media were dissected to determine the percent of viable spores. At least 20 tetrads were dissected for each replicate. (B) Suppression of hedΔR NDT80-mid pif1-md spore inviability by rad51-II3A (NH2741 RCEN). (C) Frequency of chromosome VIII nondisjunction during MI. The data for WT, hedΔR , NDT80-mid and hedΔR NDT80-mid were taken from . Additional strains used in Panel C were pif1-md (NH2685 RCEN), rrm3Δ (NH2623 RCEN), hedΔR pif1-md (NH2702 RCEN), hedΔR rrm3Δ (NH2629 RCEN), hedΔR NDT80-mid pif1-md (NH2687 RCEN), and hedΔR NDT80-mid rrm3Δ (NH2637 RCEN). Number of tetrads assayed: pif1-md (3168), rrm3Δ (1062), hedΔR pif1-md (1665), hedΔR rrm3Δ (900), hedΔR NDT80-mid pif1-md (1491) and hedΔR NDT80-mid rrm3Δ (1489). (D) Suppression of hedΔR NDT80-mid pif1-md Chromosome VIII MI nondisjunction by rad51-II3A (NH2741 RCEN). Number of tetrads assayed for hedΔR NDT80-mid pif1-md rad51-II3A (3469). (E) Steady state levels of different nuclear Pif1 proteins from the strains analyzed in Panel F. Immunoblots using protein extracts made from vegetative cells were probed with α-FLAG antibodies to detect Pif1-3FLAG. Arp7 was used as a loading control. A diploid carrying untagged pif1-m1 (pJW5-m1) was included as a control. The numbers at the bottom of each lane indicate the number of plasmids in each strain. The amount of protein was quantified and normalized to Arp7 and Pif1-m1-3FLAG using the equation. [Pif1-m1-X-3FLAG/Arp7]/[Pif1-m1-3FLAG/Arp7], where X indicates a mutation. Numbers above each lane indicate the average values for three independent replicates. (F) Complementation tests for different alleles of PIF1 . The hedΔR NDT80-mid pif1-md diploid was transformed with the vector, pRS306 (v), one copy each of pJW14 ( pif1-m1-3FLAG ) and pJW14-R3E ( pif1-m1-R3E-3FLAG ), as well as two copies pJW14-K264A ( pif1-m1-K264A-3FLAG ). Transformants were sporulated on solid medium and at least 20 tetrads were dissected per transformant. The statistical significance of differences between strains was determined using the Mann-Whitney test (* = p < .05; ** = p < .01; *** = p < .001, **** = p < .0001).

Article Snippet: The Rad51 antibody was generated by Covance Research Products (now Labcorp) in guinea pigs using the peptide QKDGNDFKFNYQGDEC.

Techniques: Western Blot, Control, Mutagenesis, Transformation Assay, Plasmid Preparation, MANN-WHITNEY

Journal: PLOS Genetics

Article Title: Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

doi: 10.1371/journal.pgen.1010407

Figure Lengend Snippet: (A) Presynaptic filament shown for one side of a DSB in WT cells. Blue lines represent the duplex of one homolog while red lines represent a chromatid from the other homolog. Dots indicate the 5’ ends of the DNA. (B) Nascent D-loop or paranemic joint. (C) Conversion to a mature D-loop by removal of Dmc1 and heteroduplex formation between the invading strand and donor strand of opposite polarity. The question mark indicates that the mechanism for how this occurs is unknown. (D) Extension of the D-loop by DNA synthesis by Polδ-PCNA followed by second end capture. (E) Double Holliday junction. (F) Presynaptic filament shown for one side of a DSB in hedΔR cells with Rad54 interacting with Rad51. (G) Nascent D-loop or paranemic joint. (H) Internal D-loop created by Rad54 removal of Rad51 and heteroduplex formation between the invading strand and donor strand of opposite polarity. (I) Removal of Dmc1 from the 3’ end of the invading strand. (J) Pif1 interacts with PCNA to facilitate DNA synthesis by unwinding the duplex to extend the D-loop allowing second end capture. (K) Double Holliday junction. Not shown is the possibility that the extended ends could be disassembled to enable noncrossover formation by synthesis dependent strand annealing.

Article Snippet: The Rad51 antibody was generated by Covance Research Products (now Labcorp) in guinea pigs using the peptide QKDGNDFKFNYQGDEC.

Techniques: DNA Synthesis