Journal: Open Biology

Article Title: The human HELQ helicase and XRN2 exoribonuclease cooperate in R-loop resolution

doi: 10.1098/rsob.240112

Figure Lengend Snippet: Synthetic oligos can be used to generate in vitro R-loops that are detectable through the S9.6 antibody and gel electrophoresis. (A) Schematic representation of the synthetic oligonucleotide substrates, RL (R-loop), dsDNA, ssRNA and ssDNA, used in the dot blot experiments (top panel). The RNA was fluorescently labelled at the 3’-end with the Cy5 fluorophore (indicated by a filled circle). The sequences of the synthetic oligonucleotides are shown in §2. Formation of R-loops was verified by PAGE (bottom panel). (B) To further validate the generation in vitro R-loops, RNase H degradation assays were carried out and products were probed with dot blots using the S9.6 antibody (1:50 diluted in RNase H buffer (NEB, M0297S)). (C) The specificity of S9.6 for R-loops in vitro compared with dsDNA, ssRNA and ssDNA was verified by dot blots. (D) The S9.6 signal (±s.e., n = 3) detected and quantified by dot blots was dependent on the concentration of R-loops, although this relationship was not linear. Lack of linearity is likely due to incomplete binding of oligonucleotides to the membrane leading to uneven loss of substrate during washing steps. (E) Treatment of R-loops with HELQ did not affect R-loops (unpaired two-tailed Student’s t ‐test showed n/s, p = 0.6850) but in the presence of HELQ and RPA the S9.6 signal decreased significantly ( ****p < 0.0001) , suggesting that HELQ resolves R-loops in the presence of RPA. Treatment with RNase H completely abolished the S9.6 signal confirming the identity of R-loops ( ****p < 0.0001). Error bars represent ±s.e. from three independent experiments ( n = 3 with four repeats per experiment). (F) A Walker A mutation (K365M) completely abolishes the HELQ ability to resolve R-loops in either the presence or absence of RPA (n/s p = 5933 and n/s p = 0.6648, respectively) whereas RPA on its own does not significantly affect R-loops (n/s p = 0.3266). Error bars represent ±s.e. from three independent experiments ( n = 3 with two repeats per experiment). (G) Control reactions with R-loops in the presence of RPA alone, HELQ alone and in the presence of RPA with AMPPNP replacing ATP in the helicase reactions. Unpaired two-tailed Student’s t -tests show that there are no significant (n/s) changes in the R-loops in these conditions (RPA alone p = 0.8596, HELQ alone p = 0.8410 and RPA+HELQ p = 0.9532). Error bars represent ±s.e. from three independent experiments ( n = 3 with two repeats per experiment). Selective dot blots are shown below the graphs in ( E) and ( G) for visual illustration. An example of a complete dot blot experiment can be seen in electronic supplementary material, figure S2.

Article Snippet: Briefly, 50 ng of M13 dsDNA (rf DNA, NEB, N4018) was incubated with 100 nM of Cascade complex comprising a crRNA targeting M13 DNA, in Helicase Buffer (20 mM Tris–HCl pH 8.0, 1 mM DTT, 100 mM NaCl and 0.1 mg ml −1 BSA) in 10 µl and incubated at 37°C for 30 min.

Techniques: In Vitro, Nucleic Acid Electrophoresis, Dot Blot, Concentration Assay, Binding Assay, Membrane, Two Tailed Test, Mutagenesis, Control

Journal: Open Biology

Article Title: The human HELQ helicase and XRN2 exoribonuclease cooperate in R-loop resolution

doi: 10.1098/rsob.240112

Figure Lengend Snippet: HELQ binds to and resolves synthetic R-loops through interaction with RPA. (A) In vitro plasmid based R-loops were generated as previously described . Supercoiled M13 dsDNA plasmid (dsM13) migrated marginally faster compared with R-loop containing supercoiled M13 dsDNA plasmid (RL). (B) Incubations of R-loop containing supercoiled M13 dsDNA (RL) plasmid with HELQ, RPA and RPA+HELQ in the absence of ATP, as indicated. Control incubations of RL and dsM13 without any proteins are also shown. RPA associates with the R-loop and produces smeared shifts characteristic of heterogeneous RPA-RL complexes while in the presence of HELQ these complexes become even more smeared due to increased heterogeneity. (C) Quality control experiments to determine the effect of EDTA and proteinase K (PK), used to remove the cascade complex after the generation of the R-loop, on the stability and electrophoretic mobility of RL and dsM13 plasmids. EDTA and PK have no effect on the stability and electrophoretic mobility of RL and dsM13 plasmids. (D) RPA binding to the R-loop in the presence of EDTA causes significant mobility shift of RL which is reversed when RPA is digested by PK. The substrate reverts to an R-loop showing that mere binding of RPA to the R-loop is not sufficient to resolve it. (E) In vitro HELQ helicase assays with a synthetic R-loop show that HELQ can resolve R-loops. The highest level of R-loop resolution was achieved when HELQ and RPA (±s.e., n = 3) were incubated with synthetic R-loops, compared with HELQ alone, which showed moderate R-loop resolution activity. RPA had no detectable effect on synthetic R-loops. (F) Schematic representation of the domain architecture of human HELQ. The N-HELQ and C-HELQ domains are shown together with the locations of important structural features; the four-helix bundle of a PWI-like fold, DEAD-box, core helicase, Winged Helix Domain (WHD) and Helix-Hairpin-Helix (HHH) elements. (G) Whisker plots showing that the C-HELQ domain, carrying the helicase core activity but lacking the RPA-interacting N-HELQ domain, is not capable of resolving R-loops. Removal of N-HELQ abrogates the HELQ interaction with RPA which is essential for the removal of R-loops (the error bars represent maximum and minimum values from three independent experiments, n = 3 with two repeats per experiment). Unpaired two-tailed Student’s t -tests show that there are no significant (n/s) changes in the RL+RPA, RL+C-HELQ and RL+RPA+C-HELQ plots relative to the R-loop (RL) alone plot ( p = 0.5941, p = 0.6226 and p = 0.2369, respectively).

Article Snippet: Briefly, 50 ng of M13 dsDNA (rf DNA, NEB, N4018) was incubated with 100 nM of Cascade complex comprising a crRNA targeting M13 DNA, in Helicase Buffer (20 mM Tris–HCl pH 8.0, 1 mM DTT, 100 mM NaCl and 0.1 mg ml −1 BSA) in 10 µl and incubated at 37°C for 30 min.

Techniques: In Vitro, Plasmid Preparation, Generated, Control, Binding Assay, Mobility Shift, Incubation, Activity Assay, Whisker Assay, Two Tailed Test

Journal: Journal of molecular biology

Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

doi: 10.1016/j.jmb.2004.07.012

Figure Lengend Snippet: Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of lambda exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.

Article Snippet: M13mp18 replicative form (RF) was purified from infected E. coli UT481 [Δ( lac-pro ) hsdS (r − m − ) lacI q lacZ ] cells using the Qiagen maxi plasmid kit.

Techniques: Incubation, Staining

Journal: Journal of molecular biology

Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

doi: 10.1016/j.jmb.2004.07.012

Figure Lengend Snippet: Non-homology at the 3′ end of the pairing strand delays strand exchange. Strand exchange was performed by UL12 and ICP8 as described in Materials and Methods with either ssM13mp18 (filled squares) or ssM13wins (open circles). The dsDNA substrates used were prepared by PCR as 32P-labeled 3.5 kb fragments using M13wins as template. Both [α-32P]dCTP and [α-32P]dATP were used for labeling of this substrate. The 1.5 kb block of HSV-1 sequence, represented by rectangles, is at the 3′ end of the pairing strand, and the remaining 2 kb are M13mp18 sequence. The wavy line/filled rectangle represents the strand complementary to the ssDNA substrates. The 3.5 kb fragment was cut with BamHI to completely remove the 1.5 kb region of non-homology, and the 2 kb fragment was purified. This substrate was used in A. The 3.5 kb fragment was also cut with EcoNI, to create a substrate possessing 170 bases of DNA non-homologous to M13mp18. The 2.17 kb fragment was purified and used for strand exchange illustrated in B. In C, the full-length 3.5 kb fragment was used, which has 1.5 kb of DNA non-homologous to M13mp18. Percentage strand exchange was calculated as the percentage of radioactivity in slowly migrating species representing strand exchange products, out of the total radioactivity in the lane. Results are the averages of two independent experiments. D, The results of the nuclease digestion of the dsDNA substrates during the course of the strand exchange assay. Results are the averages of at least three DNA samples per time-point.

Article Snippet: M13mp18 replicative form (RF) was purified from infected E. coli UT481 [Δ( lac-pro ) hsdS (r − m − ) lacI q lacZ ] cells using the Qiagen maxi plasmid kit.

Techniques: Labeling, Blocking Assay, Sequencing, Purification, Radioactivity