Genome instability due to ribonucleotide incorporation into DNA

Abstract

Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine whether ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active-site methionine in yeast DNA polymerase ϵ (Pol ϵ). Ribonucleotide incorporation in vitro was three-fold lower for M644L and 11-fold higher for M644G Pol ϵ compared to wild-type Pol ϵ. This hierarchy was recapitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S phase, had elevated dNTP pools and generated 2-5-base-pair deletions in repetitive sequences at a high rate and in a gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

Figure 1. rNMP incorporation and bypass by Pol ε derivatives

(a) Discrimination against rNMP insertion, determined as described in reference . (b) SDS-PAGE analysis of 3 μg of purified Pol ε derivatives, visualized with Coomassie Blue. (c) Stable rNMP incorporation into DNA, determined as described in reference . The marker lanes (M) depict products generated by Pol α prior to gel purification. The percentages of alkali sensitive product and the percentages of rNMP incorporation per nucleotide synthesized are shown below each lane. (d) Frequency of rNMP incorporation by M644L (blue bars), wild type (green bars), and M644G Pol ε (red bars) at each of 22 template positions. (e) Phosphorimage of rGMP bypass products for reactions incubated for 20 min. The template is shown on the right; X denotes dG or rG. The asterisks denote the position of full-length products. NE, no enzyme. (f) Insertion, extension, and relative bypass probabilities for M644L, wild type and M644G Pol ε. Values are percentages and error bars are standard deviations for three time points, calculated as described in reference .

Figure 2. rNMPs in genomic DNA of Pol ε strains ± RNase H2

(a) Alkali sensitivity of yeast genomic DNA. The marker lanes (M) are DNA fragments whose lengths in kilobase pairs are 12, 11, 10, 9, 8.1, 7.1, 6.1, 5.1, 4.1, 3.1, 2.0, 1.6, 1.0 and 0.52. (b) Cleavage of a double-stranded substrate containing a single ribonucleotide by yeast extracts. The position of the single ribonucleotide (rA) in the double-stranded oligonucleotide substrate is underlined. The 5′-end-labeled strand is indicated by and asterisk, S is substrate and P is cleavage product.

Figure 3. Characteristics of Pol ε strains ± RNase H2

(a) Growth of serial dilutions of overnight cultures plated on YPDA agar plates. (b) Growth in liquid YPDA medium, represented as the mean ± SD. (c) Flow cytometry histograms obtained as described in reference . (d) Analysis of dNTP levels, determined as described in reference . Two independent isolates were analyzed for each genotype.

Figure 4. Mutational spectra in pol2-M644G rnh201Δ strains

The coding strand of the 804 base pair URA3 open reading frame is shown. The sequence changes observed in independent ura3 mutants are depicted in red above the coding sequence for URA3 orientation 1, and in blue below the coding sequence for URA3 orientation 2. Letters indicate single base substitutions, closed triangles indicate single base additions, open triangles indicate single base deletions, and short lines above or below the coding sequence indicate 2–5 base deletions. Solid boxes enclose perfect direct repeat sequences, and dashed boxes enclose imperfect direct repeat sequences. The solid green box highlights a CACA repeat at which no deletions were observed.