Substrates, Nuclear Extracts, and MMR Assays
The substrates were generated as described previously (Baerenfaller et al., 2006
). Briefly, the hetero- and homoduplexes were constructed by primer extension using the oligonucleotides listed below as primers and the single-stranded phagemid DNA as template. Depending on the orientation of the ribonucleotide and the nick, two different ssDNA templates were used (pRichi-350topSalI creates 3′ substrates, and pRichi-2850topSalI creates 5′ substrates). After primer extension, ligation, and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, MMR assays were carried out as described (Baerenfaller et al., 2006
Unless otherwise specified, the MMR reactions were carried out with 100 ng DNA substrate and 100 μg nuclear extracts in a total volume of 25 μl in a buffer containing 20 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 110 mM KCl, 1 mM glutathione, 50 μg/ml BSA, 100 μM dNTPs, and, where indicated, 2 μCi of [α-32P]dCTP. The reactions were incubated at 37°C for 30 min. For time course experiments, 8 μl aliquots were withdrawn at the indicated time points. The reaction was stopped by adding an equal volume of 2× stop solution containing 1 mM EDTA, 3% SDS, and 5 mg/ml Proteinase K. The samples were incubated at 45°C for 30 min, purified on Mini-Clean columns (QIAGEN), and subjected to restriction digests. The digested DNA was resolved on 1% agarose gels. Nuclear extracts used in this study were prepared from HEK293, LoVo, 293TLα−, and mouse embryonal fibroblasts obtained from RNase H2, +/−, −/− mice as indicated in the figures.
All primers were obtained from Microsynth (Balgach, Switzerland). The sequences are indicated below. The SalI restriction site (GTCGAC) is underlined. The mispaired residue is highlighted in bold. The position of the ribocytidine is indicated in lower case.
T/G-SalI primer is as follows: 5′-CCAGACGTCTGTTGACGTTGGGAAGCTTGAG-3′. 5′ rC-T/G primer is as follows: 5′-GAATTGTAATAcGAACACTATAGGGCGAATTGGCGGCCGCGATCTGATCAGATCCAGACGTCTGTTGACGTTGGGAAGCTTGAG-3′. 5′-rC-C/G primer is as follows: 5’GAATTGTAATAcGAACACTATAGG-3′. 3′ rC-T/G-60 primer is as follows: 5′-CCAGACGTCTGTTGACGTTGGGAAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTcATAGCTGTTTCCTGTGTG-3′. 3′ rC-C/G primer is as follows: 5′-GCGTAATCATGGTCATAGCTGTTTCC-3′. 3′ rC-T/G-308 primer is as follows: 5′-GCTTCCTCGCTCACTGAGTCGCTGcGCTCGGTCGTTC-3′.
Antibodies and Recombinant Proteins
The RNASEH2A antibody for western blots (rabbit polyclonal, GeneTex) was used at a dilution of 1:1,000, MSH2 (mouse monoclonal, BD Transduction Laboratories) was used at a dilution of 1:1,000, and MLH1 (mouse monoclonal, Oncogene) was used at a dilution of 1:1,000. The RNase H2 (sheep polyclonal) antibody used for immunodepletion was generated by A.P.J. Recombinant MutSα and MutLα were expressed and purified in our laboratory. RNase HI and HII were obtained from New England BioLabs.
This was performed essentially as described (Cejka et al., 2005
). The oligonucleotide heteroduplexes were created by annealing 5′ radiolabelled (
) oligonucleotide 5′CTCAAGCTTCCCAACGTCG
ACAGACGTCTGG3′ with the following unlabeled oligonucleotides: T/G mismatch, 5′-CCAGACGTCTGTT
GACGTTGGGAAGCTTGAG-3′; C/G match, 5′-CCAGACGTCTGTC
GACGTTGGGAAGCTTGAG-3′; and rC/G mismatch, 5′- CCAGACGTCTGTc
GACGTTGGGAAGCTTGAG 3′, where boldface nucleotides define the residues creating the mismatches. The position of the ribocytidine is indicated in lowercase. The binding reaction mixtures contained 40 fmol oligonucleotide duplex T/G
, or rC/G
and MSH2/MSH6 (100 or 250 ng). Protein-bound substrates were separated from free probes by electrophoresis on a 6% native polyacrylamide gel eluted with TAE.
In Vitro Nicking Assay
Supercoiled DNA substrates (100 ng) were incubated with 1 U of Nt.BstNBI, RNase HI, or RNase HII in buffers recommended by the manufacturers. Nicking assays with recombinant human RNase H2 (WT or the catalytically inactive DD/AA mutant, 0.1, 1, and 10 μM) were carried out with 100 ng of supercoiled rC-T/G substrate in a buffer containing 20 mM Tris.HCl (pH 7.6), 5 mM MgCl2, 110 mM KCl, 1 mM glutathione, and 50 μg/ml BSA. The substrates were incubated at 37°C for 45 min. The products were then loaded on a 1% agarose gel and visualized with GelRed.
Mismatch-Dependent Strand Degradation Assays
The experiments were performed essentially as described (Genschel and Modrich, 2003
) with some modifications. Briefly, 100 ng of the homo-/heteroduplex was treated with 1 U of bacterial RNase HII or 10 μmole human RNase H2 (WT or DD/AA) in a reaction containing the recombinant MMR proteins (150 ng MutSα, 100 ng MutLα, 70 ng RPA, and 1.6 ng of EXO1 in 20 mM Tris.HCl [pH 7.6], 1 mM glutathione, 5 mM MgCl2
, 0.05 mg/ml BSA, 3 mM ATP, 100 mM KCl). Mismatch-provoked excision reactions were allowed to proceed for 7 min at 37°C, following which the samples were digested with HindIII and XmnI at 37°C overnight and analyzed on 1% agarose gels containing GelRed.
siRNA and Knockdown Experiments
293 cells were transfected with 40 pmol of siRNA against luciferase (siLuc), RNase H1, RNase H2a, or both H1 and H2a, using RNaiMax (Invitrogen) as per the manufacturer’s protocol. For each knockdown, six 15 cm dishes were transfected. The following day, the plates were trypsinized and scaled up into 20 15 cm dishes. Forty-eight hours posttransfection, the nuclear extracts were prepared as described above. The siRNAs used in this study were as follows: siLuc, 5′-CGUACGCGGAAUACUUCGA-3′; siRNase H1, 5′-GAAGACAAGUGCAGGGAAA-3′; and siRNase H2A, 5′-GGACUUGGAUACUGAUUAU-3′.
RNase H2-Immunodepletion of Nuclear Extracts
Protein A/G beads (SantaCruz) were washed twice with binding buffer (30 mM HEPES.KOH [pH 7.5], 7 mM MgCl2) and incubated with the RNase H2 antibody at 4°C for 3 hr (10 μl of the serum was used to bind 25 μl of the bead slurry). They were then washed thrice with the binding buffer and subsequently used to immunodeplete the nuclear extracts. For 150 μg of nuclear extracts, 10 μl of antibody-preadsorbed beads were used. Mock-depleted nuclear extracts were obtained by incubating with the beads alone. The immunodepletion was carried out for 30 min at 4°C, and the MMR assays were performed immediately.
In Vivo Mutagenesis Assays
Strand-specific mutation rates in S. cerevisiae ogg1Δ
strains were measured using the ura3-
29 marker as described previously (Pavlov et al., 2003
). The RNH201
deletions were generated by standard genetic procedures. Mutation rates were obtained by fluctuation tests using 9–20 independent cultures. The 95% confidence limits for the median and the differences between mutation frequencies using the Mann-Whitney nonparametric criterion were determined as described (Dixon and Massey, 1983
In the second assay, we used a reporter cassette containing a (T)9
repeat within the URA3
open reading frame that had been introduced into the ADE2
locus by standard genetic techniques. Mutation rates were calculated from fluctuation analysis with 32–50 independent cultures using the Lea-Coulson method of median (Lea and Coulson, 1949
), based on the appearance of mutants resistant to 5-FOA. The fluctuation analysis calculator (FALCOR) software was used to calculate the mutation rate and confidence levels (Hall et al., 2009
). The strains were derivatives of FF18733/FF18734 (Cejka et al., 2005
Genomic DNA from ten URA+ clones per S. cerevisiae ogg1Δ
strain was purified using an YDER Kit (http://www.piercenet.com/browse.cfm?fldID=06010499
). The URA3 region containing the ura3-29 mutation was amplified by PCR using the primers 5′-GAACGTGCTGCTACTCATCC-3′ and 5′-CATTCTGCTATTCTGTATAC-3′. The PCR product was sequenced by Cogentech (http://www.cogentech.it/
) using the primer 5′-TAGTTGAAGCATTAGGTCCC-3′.
Mutations flanking the T9 repeat were identified by sequencing a 500 bp fragment covering the first 242 bp of the URA3 coding sequence (including the T9 repeat), amplified by PCR using primers 5′-GCATTGGATGGTGGTAACG-3′ and 5′-GGAACGACAGTACCCTCATAAC-3′. PCRs were carried out using ReddyMix PCR Master Mix (Thermo Scientific) and 10 ng of yeast genomic DNA (95°C 5 min; then three cycles of 94°C 30 s, 65°C 30 s, and 72°C 45 s; then three cycles of 94°C 30 s, 62°C 30 s, and 72°C 45 s; then three cycles of 94°C 30 s, 59°C 30 s, and 72°C 45 s; then 35 cycles of 94°C 30 s, 56°C 30 s, and 72°C 45 s; then 72°C for 10 min). Purified PCR amplification products from 28–50 independent clones were sequenced using dye-terminator chemistry and electrophoresed on an ABI 3730 capillary sequencer (Applied Biosystems). Sequencing data were analyzed using Sequencher 4.8 (Gene Codes).