Abundant Incorporation of rNTPs into DNA Sensitizes Cells to Replication Stress and Is Lethal in Cells Lacking RNase H
(A) To test sensitivity to sublethal doses of HU or MMS, 10-fold serial dilutions of the indicated mutant strains were plated on YPD, YPD + 25 mM HU and YPD + 0.04% MMS. Pictures were taken after 3 days of incubation.
(B) Tetrads derived from a cross between rnh1Δ rnh201Δ and rnh1Δ pol2-M644G were dissected on YPD plates. Seven tetrads (1–7) are shown. The circles on the figure indicate the position of the original rhn1Δ rnh201Δ pol2-M644G spores.
(C) Sensitivity to HU and MMS of the indicated strains was tested as described in (A). A checkpoint-defective mec1-1 strain was included as a positive control.
(D) Single cells were isolated on YPD plates and grown for 22 hr in the presence of 25 mM HU; colonies were visualized by microscopic analysis.
(E and F) wild-type and rnh1Δ rn201Δ cells were released in 25 mM HU after α factor arrest. After 180 min, cultures were analyzed by FACS, for DNA contents, and cell extracts were tested by western blotting with anti-Rad53 antibodies.
(G) Wild-type and rnh1Δ rnh201Δ cells were plated on YPD with or without 25 mM HU in the presence of Phloxine B, which stains in red colonies containing dead cells.
(H) Quantification of cell survival was obtained by plating G1 synchronized cells (100 cells per plate) on dishes containing 25 mM HU or mock. Colonies were counted after 3 days of incubation. The graph is representative of three independent experiments. Error bars describe standard deviation.

Pol ζ Allows Cells to Cope with Unrepaired rNMPs
The sensitivity to HU was measured as described in Figure 1: the specific contribution of each TLS polymerase (A) and the requirement of the catalytic activity of Rev1 (B) were tested.

In the Absence of RNase H, the PRR Pathway Is Constitutively Activated and Promotes Cell Survival in an Unperturbed Cell Cycle
The role of PRR was assessed in unperturbed rnh1Δ rnh201Δ cultures. Exponentially growing cells lacking RNase H and defective in PRR were analyzed by FACS (A), to monitor cell cycle distribution, and by Phloxine B staining (B), to evaluate cell lethality. PCNA was affinity purified from exponentially growing unperturbed wild-type cells or from cells lacking RNase H activity. PCNA levels were estimated by western blotting with anti-HIS Ab. PCNA ubiquitylation was monitored by western blotting with anti-ubiquitin Ab (C), and PCNA sumoylation was monitored by western blotting with anti-SUMO Ab (D).

Postreplication Repair Is Specifically Required to Tolerate rNMPs-Containing Chromosomes
Sensitivity to sublethal doses of HU was assayed as described in Figure 1. Pictures were taken after 3 days of incubation. The contribution of NER (A), BER (A), the two branches of PRR (B), and RAD51 (D) was tested. In (C), Quantification of cell survival was obtained as described in Figure 1H. The graph is representative of three independent experiments. Error bars describe standard deviation. It is worth noting that mms2Δ TLSΔ cells, despite being sensitive to HU in the spot tests, do not exhibit increased cell lethality, suggesting that the HU sensitivity derives from a very slow cell-cycle progression. In (E), tetrads derived from a cross between rnh1Δ rnh201Δ and rad52Δ were dissected on YPD plates. Five tetrads (1–5) are shown.The circles on the figure indicate the position of the original rhn1Δ rnh201Δ rad52Δ spores. Cells derived from such microcolonies do not grow when restreaked, revealing that a rad52Δ mutation is synthetic lethal with deletion of the RNH1 and RNH201 genes. TLSΔ comprises rev1Δ rev3Δ rev7Δ rad30Δ.

DNA Polymerase ζ Efficiently Bypasses rNMPs in the Template Strand
(A) Primer-template sequences. In the 65-mer substrate, “x” is the position of the single rNMP (rG, rC, rA, or rU) and “g” is the position of the 5′-rG in the DNA template. In the 45-mer substrate, the underlined lowercase nucleotides indicate the position and sequence of the rNMPs in 4- and 16-rNMP substrates, respectively.
(B and C) Phosphorimages of products generated during bypass of a single rNMP (B) and tracts of rNMPs by Pol δ and ζ (C). The template sequence is shown on the left, the arrow depicts the location of full-length product, and “r” represents the position of the rNMPs in the template. No enzyme was added to the unextended primer reaction (0 min).
(D) Relative bypass efficiencies for Pols δ and ζ. Images of reaction products shown in (B) and (C) were quantified, and relative bypass efficiencies were calculated as described (Stone et al., 2009). The values for Pol δ with the 65-mer substrates in the absence of PCNA have been reported previously (Watt et al., 2011) and are shown here for comparison. The asterisks indicate the relative bypass values for Pol δ for reaction mixtures containing 200 nM PCNA.
(E) Mutation rates for the pol2-M644G rnh201Δ and pol2-M644G rnh201Δ rev3Δ strains. The total mutation rates for resistance to 5-FOA were determined as described in Experimental Procedures. The 95% confidence intervals for the pol2-M644G rnh201Δ and pol2-M644G rnh201Δ rev3Δ strains were 110 to 200 and 57 to 140, respectively. For the pol2-M644G rnh201Δ strain, the rates for total 2–5 base pair deletions and for CA deletions at position 216–219 in URA3 are from Clark et al. (2011). For the pol2-M644G rnh201Δ rev3Δ strain, rates for short deletions were calculated after sequencing the ura3 gene in 163 independent 5-FOA resistant clones. Of these, 136 harbored 2–5 base pair deletions, 88 of which were CA deletions at the CACA hotspot at position 216–219 in URA3 (see spectrum in Figure S6).

RNase H and Postreplication Repair Protect Cells from rNMPs Incorporated in Chromosomal DNA
During DNA synthesis, replicative polymerases can incorporate rNTPs (red dot) in place of dNTPs (A). RNase H1 and RNase H2 are required to remove rNMPs from newly replicated DNA (blue line) (B). If rNMPs persist until the following cell cycle, they will create problems at during the DNA synthesis step (C), since replicative polymerases cannot efficiently elongate the nascent strand opposite rNMPs in the template strand (black line). Replication fork restart downstream of the lesions leaves incomplete replication products for postreplication repair (D). PCNA is ubiquitylated. Either MMS2-dependent template switch mechanisms (E) or Pol ζ-dependent translesion synthesis (F) allow bypass of rNMPs and completion of replication (G). Under these conditions, inactivation of PRR causes cell lethality (H).