recombinant escherichia coli  (Cusabio)

Journal: Nature Communications

Article Title: R-loops acted on by RNase H1 influence DNA replication timing and genome stability in Leishmania

doi: 10.1038/s41467-025-56785-y

Figure Lengend Snippet: A Immunofluorescence analysis to detect R-loops in wild type cells using S9.6 antibody; -RNase H and +RNase H indicate mock or treatment with recombinant RNase HI prior to incubation with antibody, respectively; n and k , nuclear and kinetoplast DNA, respectively; image is representative of three independent experiments. B Snapshot showing DRIP-seq signal relative to the indicated features in a representative genomic region; from top to bottom: track 1 and 2 (green), R-loop enriched regions relative to input material; R-loop peaks are indicated as purple horizontal bars below track 1; track 3 (dark red), chromatin accessibility as determined by MNase-seq; track 4 (blue), G quadruplex structures (G4s) as determined by G4-seq; track 5 (purple), splice leader (SL) acceptor sites as determined by RNA-seq; track 6 (yellow), polyadenylation (Poly A) acceptor sites as determined by RNA-seq; grey arrows at the bottom indicate annotated coding sequences (CDSs); further genomic regions are shown in Supplementary Fig. and . C Metaplots showing global DRIP-seq signal around CDSs relative to chromatin accessibility, G4 localization, and SL and Poly A sites; lines indicate mean and shaded areas represent SEM. D Top enriched DNA sequences motifs found in R-loop peaks, as identified by MEME analysis; e -values for each motif are shown on top of each panel; forward and reverse indicate motifs sequences as given by top and bottom strand, respectively, of reference genome.

Article Snippet: To one of the aliquots, 5 mM MgCl 2 and 2 U of recombinant Escherichia coli RNase HI (NEB) were added.

Techniques: Immunofluorescence, Recombinant, Incubation, RNA Sequencing Assay

Journal: Nature Communications

Article Title: R-loops acted on by RNase H1 influence DNA replication timing and genome stability in Leishmania

doi: 10.1038/s41467-025-56785-y

Figure Lengend Snippet: A Colourmap showing distribution of DRIP-seq signal in all 36 L.major chromosomes; chromosomes are ordered by size; -RNase H and +RNase H indicate mock or treatment with recombinant RNase HI prior to immunoprecipitation, respectively; shuffled, indicates DRIP-seq signal plotted after R-loops peaks were randomly distributed throughout the genome; an independent experiment is shown in Supplementary Fig. . B – G Colourmaps showing distribution patterns of DNA replication timing predicted by MFA-seq, putative origins of DNA replication (ORIs) predicted by SNS-seq, chromatin accessibility determined by MNase-seq, G-quadruplexes (G4s) density determined by G4-seq, distribution of directed and inverted s hort i nterspersed de generate r etroposons 1 (SIDER1) and GC fraction, respectively; chromosome 31, which does not follow the pattern of all other chromsomes for ( A , C and D ), is indicated. H – N Simple linear regression analysis showing correlation between chromosome size and chromosome-averaged signals of DRIP-seq, DNA replication timing, ORIs, chromatin accessibility, G4s, SIDER1 sequences and GC fraction, respectively. O – T Simple linear regression analysis showing correlation between averaged DRIP-seq signal at each chromosome and averaged signals of DNA replication timing, ORIs, chromatin accessibility, G4s and SIDER sequences, respectively. In panels H to T , R and P values are indicated at the top of each panel, circles indicate mean, lines indicate the best fit and shaded areas represent 95% CI. Source data are provided as a Source Data file.

Article Snippet: To one of the aliquots, 5 mM MgCl 2 and 2 U of recombinant Escherichia coli RNase HI (NEB) were added.

Techniques: Recombinant, Immunoprecipitation

Journal: Nature Communications

Article Title: R-loops acted on by RNase H1 influence DNA replication timing and genome stability in Leishmania

doi: 10.1038/s41467-025-56785-y

Figure Lengend Snippet: A Immunofluorescence analysis to detect RNase H1-HA using anti-HA antibody; cells undergoing DNA replication are shown by EdU signal; n and k indicate nuclear and kinetoplast DNA, respectively; image is representative of two independent experiments. B Line scan, plotting the RNase H1-HA and EdU signal intensity values across the dotted white line in ( A ). C RNase H1-HA versus EdU signal intensity plotted as a 2D density plot using hexagonal bins; R and P values for linear regression analysis is shown at the top. D Representative snapshot of RNase H1-HA ChIP-seq; from top to bottom: track 1 and 2 (green), DRIP-seq signal where -RNase H and +RNase H indicate mock or treatment with recombinant RNase HI prior to immunoprecipitation, respectively; track 3 (dark green), enriched regions of acetylated Histone H3 (AcH3); track 4 (dark red), β-D-glucosyl-hydroxymethyluracil (Base J) enriched regions; track 5 (dark grey), RNase H1-HA enriched regions relative to input material; grey arrows at the bottom indicate the position and orientation of polycistronic transcription units (PTUs). E Metaplots (top) and colourmap (bottom) showing RNase H1-HA ChIP-seq signal around convergent, divergent and head-to-tail strand switch regions (SSR-Conv, SSR-Div and HT, respectively); metaplots for DRIP-seq, AcH3 and Base J are also shown; in metaplots above colourmaps, lines and shaded areas represent mean and SEM, respectively. F Metaplot showing global RNase H1-HA ChIP-seq signal (dark gray) around CDSs compared with DRIP-seq signal (green); lines indicate mean and shaded areas represent SEM. G Metaplots (top) and colourmap (bottom) showing RNase H1-HA ChIP-seq signal around DRIP-seq peaks; regions were grouped using k-means clustering; percentages indicate the proportion of peaks in each cluster. H DRIP-seq, MNase-seq and SNS-seq signals were plotted around DRIP-seq peaks grouped in clusters 1 and 2 from ( G ) and represented as metaplots (top) and colourmaps (bottom). In metaplots above colourmaps in ( G and H ), lines and shaded areas represent mean and SEM, respectively. I Simple linear regression analysis showing correlation between averaged signals of RNase H1-HA ChIP-seq and DRIP-seq at each chromosome. J Simple linear regression analysis showing correlation between chromosomes length and averaged signals of RNase H1-HA ChIP-seq at each chromosome in unperturbed exponentially growing cells (NT) and after release from synchronization with hydroxyurea (HU); cell cycle progression analysis upon HU synchronization is shown in Supplementary Fig. . In panels I and J , R and P values are indicated at the top of each panel, circles indicate mean, lines indicate the best fit and shaded areas represent 95% CI. Source data are provided as a Source Data file.

Article Snippet: To one of the aliquots, 5 mM MgCl 2 and 2 U of recombinant Escherichia coli RNase HI (NEB) were added.

Techniques: Immunofluorescence, ChIP-sequencing, Recombinant, Immunoprecipitation

Journal: Nature Communications

Article Title: R-loops acted on by RNase H1 influence DNA replication timing and genome stability in Leishmania

doi: 10.1038/s41467-025-56785-y

Figure Lengend Snippet: A Schematic representation of the DiCre-mediated RNase H1 gene deletion strategy; CRISPR-Cas9 was used to flank the RNase H1 ORF with LoxP sites and fuse it with an HA tag ( RNase H1-HA Flox ); rapamycin-mediated activation of DiCre was used to catalyze excision of RNase H1-HA Flox ; refer to Supplementary Fig. for the rapamycin induction strategy; a and b , annealing position of primers used in ( D ). B Western blotting analysis of whole cell extracts from RNase H1-HA Flox cells ~48 h after growth in the absence (−RAP) or in the presence (+RAP) of rapamycin at passages 1 and 2 (P1 and P2, as shown in ( C )); extracts were probed with anti-HA antibody and anti-EF1α was used as loading control. C Growth profile of the RNase H1-HA Flox cell line cultivated in the absence (−RAP, black) or presence (+RAP, grey) of rapamycin; cells were seeded at ~10 5 cells.mL −1 at day 0 and diluted back to that density every 4–5 days for seven passages (P1 to P7); cell density was assessed every 24 h in two independent experiments. D PCR analysis of genomic DNA extracted from RNase H1-HA Flox cells ~48 h in the indicated passages, after growth in the absence (−RAP) or in the presence (+RAP) of rapamycin; annealing positions for primers a and b are shown in ( A ); image is representative of two independent experiments. E Growth profile of a clonal RNase H1 KO cell line, selected after DiCre-mediated RNase H1 gene deletion compared to wild type (WT) cells ; cell density was assessed every 24 h and is represented as the mean from four independent experiments; error bars indicate SEM. F Immunofluorescence analysis using S9.6 antibody to detect R-loops with (+HU) or without (−HU) 5 mM hydroxyurea treatment for 6 h. G Quantification of R-loops levels detected via immunofluorescence using S6.9 antibody in the indicated conditions, represented as arbitrary units (arb. units); −RNase H and +RNase H indicate mock or treatment with recombinant RNAse HI prior to incubation with antibody, respectively; quantification is representative of three independent experiments; (****),(**) and ns: p < 0.0001, p = 0.0089 and not significant, respectively, as determined by Kruskal–Wallis test (one-way ANOVA) using Dunn’s test for multiple comparison correction. H Representative snapshot showing DNA replication timing on the entire chromosome 22, as determined by MFA-seq using exponentially growing cells normalized with stationary cells; positive and negative values indicate early and late replicating regions, respectively; grey arrows at the bottom indicate the position and orientation of polycistronic transcription units (PTUs). I , J Metaplots showing global MFA-seq signal in early and late replicating regions, respectively; lines indicate mean and shaded areas represent SEM; (****),(**), (*) and ns: p < 0.0001, p = 0.0054, p = 0.0188 and not significant, respectively, as determined by Kruskal–Wallis test (one-way ANOVA) using Dunn’s test for multiple comparison correction. K Simple linear regression analysis showing correlation between chromosome size and averaged MFA-seq signal; R and P values are indicated at the top of each panel, circles indicate mean, lines indicate the best fit and shaded areas represent 95% CI. Source data are provided as a Source Data file.

Article Snippet: To one of the aliquots, 5 mM MgCl 2 and 2 U of recombinant Escherichia coli RNase HI (NEB) were added.

Techniques: CRISPR, Activation Assay, Western Blot, Control, Immunofluorescence, Recombinant, Incubation, Comparison

Journal: Nature Communications

Article Title: R-loops acted on by RNase H1 influence DNA replication timing and genome stability in Leishmania

doi: 10.1038/s41467-025-56785-y

Figure Lengend Snippet: A Schematic representation showing time points from which cells were collected for whole genome sequencing (WGS). B Colourmap showing genome-wide relative copy number variation (CNV) analysis; chromosomes are ordered by size from top to bottom; CNV in RNase H1-HA Flox cells is expressed as log 2 [ratio(normalized reads from P7/normalized reads from P4)] for either –RAP or +RAP conditions; CNV in KO cell line is expressed as log 2 [ratio(normalized reads from KO/ normalized reads from P4 -RAP)]; chromosomes 05 and 12, showing decreased copy number, are indicated. C Metaplots showing averaged CNV profiles across all chromosomes (grey); averaged DRIP-seq profiles across all chromosomes from 2A is also shown at the top (green). D Metaplots (top) and colourmap (bottom) showing relative CNV profiles around DRIP-seq peaks upon k-means clustering from 3G. In metaplots from C and D, lines indicate mean and shaded areas represent SEM. E Relative CNV analysis at the indicated loci ; CNV is expressed as in ( B ); DRIP-seq (green) and RNase H1-HA ChIP-seq (black) signals are also shown at the top. F Absolute chromosome CNV analysis for the indicated chromosomes in the indicated conditions and passages; violin plots represent the distribution of normalized read counts relative to the haploid genome content; (****),(***) and (*): p < 0.0001, p = 0.0005 and p = 0.0141, respectively, as determined by Kruskal-Wallis test (one-way ANOVA) using Dunn’s test for multiple comparison correction. Source data are provided as a Source Data file.

Article Snippet: To one of the aliquots, 5 mM MgCl 2 and 2 U of recombinant Escherichia coli RNase HI (NEB) were added.

Techniques: Sequencing, Genome Wide, ChIP-sequencing, Comparison

Journal: Nature Communications

Article Title: R-loops acted on by RNase H1 influence DNA replication timing and genome stability in Leishmania

doi: 10.1038/s41467-025-56785-y

Figure Lengend Snippet: A , D Metaplots (top) and colourmaps (bottom) showing normalized density of new SNPs and InDels, respectively, around annotated coding sequences (CDSs). B , E Metaplots (top) and colourmaps (bottom) showing normalized density of new SNPs and InDels, respectively, around DRIP-seq peaks; regions were grouped using k-means clustering; percentages indicate the proportion of peaks in each cluster. C , F Metaplots of DRIP-seq, RNase H1-HA ChIP-seq, MNase-seq and SNS-seq signals around DRIP-seq peaks grouped in clusters 1, 2 and 3 from B and E, respectively. In metaplots from ( A to F ), lines indicate mean and shaded areas represent SEM. G , H SNP and InDel densities in chromosomes grouped by length; smaller: 0.268–0.622 Mb, medium: 0.629–0.840 Mb, larger: 0.913–2.68 Mb; (****), (***), (**), (*) and ns: p < 0.0001, p = 0.0001, p = 0.0012, p = 0.0155 and not significant, respectively, as determined by one-way ANOVA and Fisher’s LSD test. Source data are provided as a Source Data file.

Article Snippet: To one of the aliquots, 5 mM MgCl 2 and 2 U of recombinant Escherichia coli RNase HI (NEB) were added.

Techniques: ChIP-sequencing