Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis
Summary
Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.
Abstract
The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.
Introduction
During eukaryotic nuclear DNA replication, ribonucleotides are incorporated into the genome by all three major DNA replicases, DNA polymerases (Pols) α, ε, and δ1,2. RNase H2-dependent ribonucleotide excision repair (RER3) removes the majority of these embedded ribonucleotides.
A ribonucleotide in DNA is susceptible to hydrolysis, as the 2' hydroxyl group of the sugar moiety can attack the adjacent phosphodiester bond, releasing one end with a 2'-3' cyclic phosphate and the other with a 5'-OH4. Alkaline conditions can greatly accelerate this reaction. Thus, the hydrolysis of embedded ribonucleotides during incubation in a basic solution causes fragmentation of genomic DNA, which can be visualized by alkaline-agarose electrophoresis5. This DNA can be transferred to a membrane and probed by Southern blot analysis using strand-specific probes that allow the identification of alkali-sensitive sites caused by ribonucleotides incorporated into the nascent leading- or lagging-strand DNA, respectively.
In yeast cells lacking RNase H2 activity, removal of ribonucleotides can be initiated when topoisomerase I (Top1) nicks the DNA at the embedded ribonucleotide6,7. However, when Top1 cleaves on the 3' side of the ribonucleotide, this generates 5'-OH and 2'-3' cyclic phosphate DNA ends that are refractory to religation. Failure to repair, or aberrant processing of these 'dirty ends' can lead to genomic instability. In addition, if the incision occurs within a repeat DNA sequence, the repair process can lead to deletion mutations. This is particularly problematic for tandem repeats, where short deletions (of between two and five base pairs) are commonly observed in RNase H2-deficient cells. The Top1-dependent deleterious effects in the absence of yeast RNase H2 activity are exacerbated in a DNA polymerase ε mutant (pol2-M644G) promiscuous for ribonucleotide incorporation during nascent leading strand synthesis.
Processing of ribonucleotides in DNA leads to spontaneous mutations and this mutagenesis can be detected by using appropriate reporter genes and selecting for the accompanying phenotypic change. A fluctuation test or Luria and Delbrück experiment is one of the most commonly used methods to measure spontaneous mutation rates using selectable reporter genes8,9. In yeast, the URA3 and CAN1 genes can be used as reporters in a forward mutation assay, which allows for the detection of all mutation types that result in the loss of gene function. The spontaneous mutation rate is estimated as the median of that observed for multiple parallel cultures started from single colonies without mutations in the target reporter gene. A yeast RNase H2-deficient strain such as rnh201Δ has a moderately elevated overall spontaneous mutation rate that is largely caused by an elevated incidence of 2 – 5 bp deletions in tandem repeat sequences. Thus, to fully characterize the mutagenic effects of ribonucleotides in the genome, specific mutation rates need to be determined. In this case, the URA3 or CAN1 reporter genes can be amplified and sequenced to determine the types and locations of the mutations, and specific mutation rates can be calculated. Compiling mutations identified in multiple independent URA3 or CAN1 mutants can then be used to generate a mutation spectrum.
Protocol
Representative Results
Discussion
Here, we describe the protocols for two sets of experiments that are frequently used to semi-quantitatively analyze ribonucleotides incorporated during DNA replication and the mutagenic effects of unrepaired ribonucleotides. Although these approaches involve the model eukaryote S. cerevisiae, these techniques can be easily adapted to other microbes and even higher eukaryotes.
Probing for unrepaired ribonucleotides in DNA using alkaline-agarose electrophoresis coupled with Southern blo…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
We thank all current and former Kunkel Lab members for their work and discussions related to protocol and reused data presented here. This work was supported by Project Z01 ES065070 to T.A.K. from the Division of Intramural Research of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS).
Materials
| YPD media | 20 g dextrose, 20 g peptone, 10g yeast extract, in deionized H2O up to 1 L, add 20 g Bacto agar for solid media, autoclave. | ||
| COM plates | 1.3 g SC dropout mix, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar deionized H2O up to 1 L. Adjust pH to 5.8. Autoclave and 30 – 35 ml per plate. | ||
| CAN plates | 1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 1 L. Autoclave for 15 mins at 121 °C and cool down to 56 °C. Add 6 mL of filter-sterilized 1% canavanine sulfate solution. | ||
| 5-FOA plates | 1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 800 mL. Autoclave for 15 mins at 121 °C and cool down to 60 °C. Add 200 mL of filter-sterilized 0.5% 5-FOA solution. | ||
| L-Canavanine sulfate | US Biological | C1050 | |
| 5-FOA | US Biological | F5050 | |
| 20 mL glass culture tube | Any brand | ||
| Culture rotator in 30 °C incubator | Shaker incubator can be used instead | ||
| 96 well round bottom plates | Sterile, any brand | ||
| 3 mm glass beads | Fisher Scientific | 11-312A | Autoclave before use |
| 12-channel pipettes | Any brand | ||
| Ex Taq DNA Polymerase | TaKaRa | RR001 | |
| Epicentre MasterPure Yeast DNA Purification Kit | Epicenter | MPY80200 | |
| 3 M sodium acetate | Sigma-Aldrich | S7899 | |
| 100% ethanol | |||
| Qubit 2.0 Fluorometer | Invitrogen | Q32866 | Newer model available |
| dsDNA BR Assay kit | Invitrogen | Q32850 | |
| KOH | Sigma-Aldrich | 221473 | |
| EDTA | Sigma-Aldrich | E7889 | |
| Ficoll 400 | Dot Scientific Inc. | DSF10400 | |
| Bromocresol green | Eastman | 6356 | |
| Xylene cyanol FF | International Technologies Inc. | 72120 | |
| NaOH | Sigma-Aldrich | S8045 | |
| 1 M Tris-HCl (pH 8.0) | Teknova | T5080 | |
| SYBR Gold Nucleic Acid Gel Stain | Invitrogen | S11494 | |
| UV transilluminator | |||
| Amersham Nylon membrane Hybond-N+ | GE Healthcare | RPN303B | |
| 3 MM CHR Chromotography paper | Whatman | 3030-392 | |
| NaCl | Caledon | 7560-1 | |
| Stratalinker 1800 | Stratagene | ||
| QIAquick PCR Purification Kit | Qiagen | 28106 | |
| G-25 spin column | GE Healthcare | 27-5325-01 | |
| 1 M Sodium phosphate buffer (pH 7.2) | Sigma-Aldrich | NaH2PO4 (S9638); Na2HPO4 (S9390) |
|
| SDS | Sigma-Aldrich | L4522 | |
| BSA | Sigma-Aldrich | A3059 | |
| Formamide | Sigma-Aldrich | 47671 | |
| Geiger counter |
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