RNase H2-initiated ribonucleotide excision repair

Abstract

Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. Here, we present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. RER is most efficient when the ribonucleotide is incised by RNase H2, and further excised by the flap endonuclease FEN1 with strand displacement synthesis carried out by DNA polymerase δ, the PCNA clamp, its loader RFC, and completed by DNA ligase I. We observed partial redundancy for several of the enzymes in this pathway. Exo1 substitutes for FEN1 and Pol ε for Pol δ with reasonable efficiency. However, RNase H1 fails to substitute for RNase H2 in the incision step of RER.

Fig. 1. Ribonucleotide incorporation during DNA synthesis

(A) Primed RPA-coated SS-M13mp18 DNA was fully replicated by either Pol δ or Pol ε with PCNA under standard conditions, with or without rNTPs (Materials and Methods). Replication products were alkali-treated and separated on an alkaline agarose gel. (B) The radioactivity distribution was scanned and divided by the DNA size distribution to obtain a normalized molar product distribution (See Supplemental Materials for details). (C) The standard assay (lane 3) was modified in lane 1 to omit PCNA and RFC, reduce salt to 20 mM NaCl, increase Pol δ from 3 nM to 500 nM, and the incubation time from 8 min to 3 h. In lane 2 the standard assay was modified to reduce salt to 20 mM. See also Fig. S1.

Fig. 2. Ribonucleotide Excision Repair Assay

Primed RPA-coated SS-M13mp18 DNA was fully replicated by Pol δ with PCNA under standard conditions with or without rNTPs. After 8 min at 30 °C, RNase H2, FEN1, and DNA ligase I were added as indicated, and incubation continued for 4 min. Each reaction was split in two, and one half was separated on a 1% agarose gel in the presence of 0.5 μg/ml of ethidium bromide; the other half was treated with 0.3 M NaOH. The products were recovered by ethanol precipitation and separated on a 1% alkaline agarose gel. Migration positions of nicked circles and covalently closed circles on the neutral agarose gel (top panel) are indicated. Migration positions of circular ssDNA (a), linear ssDNA (b), and denatured forms of dsDNA (c) on the alkaline agarose gel (bottom panel) are indicated. See also Fig. S2.

Fig. 3. Factor requirements and redundancies in RER

Primed RPA-coated SS-M13mp18 DNA was fully replicated by Pol δ with PCNA under standard conditions with rNTPs for 8 min. In panels (A) and (D), the replicated DNA was isolated and purified, and the DNA incubated in a second reaction with: (A) RPA, RFC, Pol δ, RNase H2 or RNase H2-ΔPIP, FEN1, DNA ligase I, and the indicated concentrations of PCNA for the times indicated; (D) RPA, PCNA, RFC, RNase H2, FEN1, DNA ligase I, and either Pol δ or Pol ε for the indicated times. In panels (B) and (C), the replication reaction was supplemented with: (B) FEN1, DNA ligase I, and the indicated concentrations of either RNase H1 or RNase H2 for 10 min; (C) RNase H2, DNA ligase I, and either no nuclease, FEN1, Exo1, or Dna2 for the times indicated. The products were treated with alkali and separated on an 1% alkaline agarose gel. In (A), the median product distribution was determined as in Fig. 1B and Supplemental Materials. See also Fig. S3. The standard error for two independent experiments is given.

Fig. 4. Model for Ribonucleotide Excision Repair (RER)

Redundant functions of FEN1 with Exo1 and Pol δ with Pol ε are indicated. See text for details.