Figure 1.

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.