Measuring ribonucleotide incorporation into DNA in vitro and in vivo

Abstract

Ribonucleotides are incorporated into genomes by DNA polymerases, they can be removed, and if not removed, they can have deleterious and beneficial consequences. Here, we describe an assay to quantify stable ribonucleotide incorporation by DNA polymerases in vitro, and an assay to probe for ribonucleotides in each of the two DNA strands of the yeast nuclear genome.

Fig. 1

(a) Sequence of primer-templates used for reactions in panel b. (b) Stable rNMP incorporation. Lane marked (U) depicts products generated by Pol ε prior to gel purification. (−) indicates KCl treatment and (+) indicates KOH treatment. The percentage of alkali-sensitive product and the percentage of rNMP incorporation per nucleotide synthesized is shown below the lane. (c) Percentage of rNMP incorporation by Pol ε at each of 24 template positions. The position and identity of each incorporated rNMP are displayed on the Y-axis

Fig. 2

An example of an alkaline agarose gel stained with ethidium bromide. All strains in this experiment harbor an M644G variant of the leading strand replicase, Pol ε, encoded by the POL2 gene. This pol2-M644G variant has increased capacity to incorporate ribonucleotides in vitro and in vivo [8, 12, 13]. Alkali-sensitive sites in the nuclear genome of rnh201Δ strains (lanes designated (−)) that are deficient in RNase H2 activity indicate the presence of unrepaired ribonucleotides

Fig. 3

(a) Schematic diagram of the orientation of the URA3 reporter gene on chromosome III adjacent to the ARS306 origin of replication. Template strands are in black, the nascent leading strand is in blue and the nascent lagging strand in green. The orientation of the reporter gene with respect to coding sequence is indicated as orientation 1 (OR1) or orientation 2 (OR2). Strand-specific radiolabeled probes that anneal to one of the two nascent strands are designated Probe A and Probe B. Their strand-specificity is dependent on the orientation of URA3. (b) An example of a southern blot probing for alkali-sensitive sites in the nascent leading and lagging strands of yeast genomic DNA. All strains in this experiment harbor the pol2-M644G variant that has increased capacity to incorporate ribonucleotides [8, 12, 13]