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 δ^1,2. 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 c....

Protocol

1. Alkaline Hydrolysis and Strand-specific Southern Blot (Figure 1)

1. Cell growth and genomic DNA isolation
   1. Isolate yeast genomic DNA using a yeast DNA purification kit following the manufacturer’s instructions. Use the cultures that are in the exponential phase of growth (between an optical density of 0.5 and 1). Resuspend the final DNA pellet in 35 mL of 1x TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA)).
   2. Add 1 mL of 5 mg/mL RNase A to the DNA and incubate for 30 min at 37 °C.
   3. Precipitate the DNA using 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.....

Results

Treatment of genomic DNA with alkali followed by alkaline gel electrophoresis allows for semi-quantitative detection of the DNA fragmentation due to the abundance of stably incorporated ribonucleotides. Figure 2 shows the gel images of yeast genomic DNA treated with or without KOH⁵. The M644L variant of Pol2, the catalytic subunit of Polε, has reduced the ability to incorporate ribonucleotides while the M644G mutant incorporates more r.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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