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Rnase h2

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RNase H2 is a recombinant ribonuclease H2 enzyme that cleaves the RNA strand in RNA-DNA hybrids. It is a multi-subunit enzyme that recognizes and cleaves the RNA strand in RNA-DNA hybrid structures.

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Lab products found in correlation

Taq dna polymerase, by New England Biolabs (2 mentions) Rnase h1, by Thermo Fisher Scientific (2 mentions) Prime it 2 random primer labeling kit, by Agilent Technologies (1 mentions) Typhoon laser scanner, by GE Healthcare (1 mentions) Phosphoimager screen, by GE Healthcare (1 mentions) Hybond n membrane, by Cytiva (1 mentions) T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions) Typhoon 9500 imager, by GE Healthcare (1 mentions)

6 protocols using rnase h2

1

Mitochondrial DNA Structural Analysis

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1 μg of isolated mtDNA was linearized with SacI, precipitated and dissolved in 10 mM Tris HCl pH 7.5. The DNA was hydrolyzed with 0.3 M NaOH at 55°C or digested with RNase H2 (New England Biolabs) at 37°C for 2 h. Samples were run on a 0.8% agarose alkaline gel (30 mM NaOH, 1 mM EDTA) at 25 V, 4°C for 20 h and blotted onto Hybond-N+ membrane (Amersham, GE Healthcare). Single-stranded probes were end-labelled with γ-32P ATP using T4 polynucleotide kinase (Thermo Scientific) following the manufacturer’s protocol. Double-stranded probes were generated by labelling an approximately 500 bp PCR product with α-32P dCTP using Prime-It II Random Primer Labeling kit (Agilent Technologies). Hybridization was for 16 h at 42°C for ssDNA probes and at 65°C for dsDNA probes. The membrane was exposed to a PhosphoImager screen and scanned in a Typhoon laser scanner (GE Healthcare). The radioactive intensity was quantified using ImageJ software and plotted on a distribution plot. The median size of alkali-treated products was determined from the distribution of the radioactivity intensity and related to the size marker that was run in parallel [7 (link)].
Forslund J.M., Pfeiffer A., Stojkovič G., Wanrooij P.H, & Wanrooij S. (2018). The presence of rNTPs decreases the speed of mitochondrial DNA replication. PLoS Genetics, 14(3), e1007315.
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2

DNA Replication Initiation Analysis

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Isolated DNA (1.8 μg) was incubated for 1 hour at 37°C with or without RNase H2 (NEB). Primer extension was performed with 2 U Taq DNA polymerase (NEB) in 1X ThermoPol buffer, 200 μM dNTPs and 1.5 pmol labeled primer. The primers were 5´-end labeled with PNK (NEB) and γ-[32P]ATP and were corresponding to L-strand positions 8-29 (GGT CTA TCA CCC TAT TAA CCA C) and 16,331-16,351 (CAC ACA TCA ACT GCA ACT CCA). The primer extension reaction was performed with 5 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 58°C, 45 seconds at 72°C and 5 minutes at 72°C with step 2-4 repeated in 20 cycles. The reactions were stopped and ethanol precipitated as for replication initiation reactions. The sequencing ladders were prepared with USB Sequenase Version 2.0 (Affymetrix) according to the manufacturers protocol. The primer extension experiment was performed multiple times with similar results and the figure shows a representative gel image for that experiment.
Posse V., Al-Behadili A., Uhler J.P., Clausen A.R., Reyes A., Zeviani M., Falkenberg M, & Gustafsson C.M. (2019). RNase H1 directs origin-specific initiation of DNA replication in human mitochondria. PLoS Genetics, 15(1), e1007781.
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3

TERRA and TelDNA Binding to RAD51

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Fluorescently labelled 41mer TERRA or 41mer TelDNA (50 nM) were pre-incubated for 10 minutes at 37 °C with increasing concentration of RAD51 in 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2 supplemented with 1 mM CaCl2 and 1 mM AMP-PNP. Reaction was started by addition of 600 ng of pCR4-TOPO vector containing the 15q subtelomeric sequence followed by 15 copies of TTAGGG repeats. The mixture was incubated for another 10 minutes at 37 °C and then stopped by addition of SDS (0,1%) and proteinase K (0.1 mg/ml) followed by 3 minutes incubations at 37 °C. Reaction mixtures were separated on 0.8% agarose gel and analyzed as described for EMSA. For digestion of R-loops by RNaseH, the mixtures were inhibited for 1 minute with PMSF (2 mM) and with EGTA (1.6 mM) at 37°C to inhibit proteinase K and chelate calcium ions, respectively. The products were digested by RNaseH1 (6.8 mU/μl, Thermo Scientific™) or RNaseH2 (6.8 mU/μl, New England Biolabs) for 1 minute at 37 °C and resolved as described above. For antibody-specific supershift of R-loops, the products were formed as described for RNaseH digestion followed by incubation with S9.6 antibodies (0.02 μg/μl) for 2 minutes at 37°C and/or anti-mouse horseradish peroxidase conjugated antibody
Feretzaki M., Pospíšilová M., Fernandes R.V., Lunardi T., Krejci L, & Lingner J. (2020). RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops. Nature, 587(7833), 303-308.
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4

TERRA and TelDNA Binding to RAD51

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Fluorescently labelled 41mer TERRA or 41mer TelDNA (50 nM) were pre-incubated for 10 minutes at 37 °C with increasing concentration of RAD51 in 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2 supplemented with 1 mM CaCl2 and 1 mM AMP-PNP. Reaction was started by addition of 600 ng of pCR4-TOPO vector containing the 15q subtelomeric sequence followed by 15 copies of TTAGGG repeats. The mixture was incubated for another 10 minutes at 37 °C and then stopped by addition of SDS (0,1%) and proteinase K (0.1 mg/ml) followed by 3 minutes incubations at 37 °C. Reaction mixtures were separated on 0.8% agarose gel and analyzed as described for EMSA. For digestion of R-loops by RNaseH, the mixtures were inhibited for 1 minute with PMSF (2 mM) and with EGTA (1.6 mM) at 37°C to inhibit proteinase K and chelate calcium ions, respectively. The products were digested by RNaseH1 (6.8 mU/μl, Thermo Scientific™) or RNaseH2 (6.8 mU/μl, New England Biolabs) for 1 minute at 37 °C and resolved as described above. For antibody-specific supershift of R-loops, the products were formed as described for RNaseH digestion followed by incubation with S9.6 antibodies (0.02 μg/μl) for 2 minutes at 37°C and/or anti-mouse horseradish peroxidase conjugated antibody
Feretzaki M., Pospíšilová M., Fernandes R.V., Lunardi T., Krejci L, & Lingner J. (2020). RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops. Nature, 587(7833), 303-308.
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5

Nuclease Assay with PCNA Regulation

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2.5 μL of XlAPE2, as a 200 nM stock in storage buffer (5
mM Tris pH 8.0, 75 mM NaCl, 0.5 mM TCEP, 0.5 mM MgCl2, 50% glycerol), or
2.5 μL of 1 U/μL APE1 (NEB) was added to 2.5 μL 200
nM substrate, 1 uL 10x reaction buffer (100 mM Tris pH 8.0, 10 mM TCEP,
10 mM MnCl2, 1 mg/mL BSA) and 4 μL ddH2O at
37°C to initiate a reaction with final concentrations of 50 nM
XlAPE2 and 50 nM substrate. For nuclease assays in the presence of PCNA,
reactions were supplemented with 50 nM PCNA (Figure S4B). At noted
timepoints, 10 μL reactions were stopped by the addition of 20
μL of formamide buffer (98% formamide, 10 mM EDTA) and immediate
denaturation at 95°C for 5 minutes. Samples were resolved on 20%
polyacrylamide TBE-Urea gels, imaged on a Typhoon 9500 imager (GE
Healthcare), and viewed using ImageJ. For RNase H2 (NEB) reactions, 2
U/μL enzyme was used with NEB-supplied 10x ThermoPol reaction
buffer (200 mM Tris-HCl pH 8.8, 100 mM
(NH4)2SO4, 100 mM KCl, 20 mM MgSO4,
1% Triton X-100).
Álvarez-Quilón A., Wojtaszek J.L., Mathieu M.C., Patel T., Appel C.D., Hustedt N., Rossi S.E., Wallace B.D., Setiaputra D., Adam S., Ohashi Y., Melo H., Cho T., Gervais C., Muñoz I.M., Grazzini E., Young J.T., Rouse J., Zinda M., Williams R.S, & Durocher D. (2020). Endogenous DNA 3′ blocks are vulnerabilities for BRCA1 and BRCA2 deficiency and are reversed by the APE2 nuclease. Molecular cell, 78(6), 1152-1165.e8.
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6

Mouse heart DNA extraction and analysis

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Total DNA was isolated from the mouse heart tissue with the Gentra Puregene Tissue Kit without RNase A treatment. An 3 μg aliquot of DNA was incubated for 1 h at 37°C with or without RNase H2 (NEB). Primer extension was performed with 2 U Taq DNA polymerase (NEB) in 1× ThermoPol buffer, 200 μM dNTPs and 1.5 pmol labelled primer. The primer, corresponding to nt 15 181–15 201, was 5′-end labelled with γ-[32P]-ATP using PNK (NEB). The primer extension reaction was initiated for 5 min at 95°C and then continued for 25 cycles with 30 s at 95°C, 30 s at 58°C, 45 s at 72°C and 5 min at 72°C. The reactions were terminated and precipitated with ethanol.
Misic J., Milenkovic D., Al-Behadili A., Xie X., Jiang M., Jiang S., Filograna R., Koolmeister C., Siira S.J., Jenninger L., Filipovska A., Clausen A.R., Caporali L., Valentino M.L., La Morgia C., Carelli V., Nicholls T.J., Wredenberg A., Falkenberg M, & Larsson N.G. (2022). Mammalian RNase H1 directs RNA primer formation for mtDNA replication initiation and is also necessary for mtDNA replication completion. Nucleic Acids Research, 50(15), 8749-8766.
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