Fig 1. MCK1 is indispensable for dun1Δ cells dealing with replication stress.

(A) Regulation of cellular dNTP levels by the S phase checkpoint in S. cerevisiae. (B) 5-fold serial dilutions of WT, mec1Δsml1Δ, rad53Δsml1Δ, chk1Δ, dun1Δ, mck1Δ, dun1Δmck1Δ (S1 Table) cells were spotted onto YPD plates supplemented with the indicated concentrations of HU. Plates were incubated at 30°C for 48 h before photograph. (C) Overexpression of MCK1 rescues the lethality of mec1Δ or rad53Δ. 5-fold serial dilutions of the indicated strains were spotted onto SC-URA or SC+5FOA (5-fluoroorotic acid) plates and grown at 30°C for 48 h. The plasmid expressing WT MCK1/URA3 was lost in the presence of 5-FOA. (D) Physical interaction between Mck1 and Rad53. MCK1 and RAD53 were cloned into pGADT7 and pGBKT7 vectors, respectively. AH109 strains transformed with the indicated plasmids were grown on the SC-Trp-Leu or SC-Trp-Leu-His plates. 0.5 mM 3-amino-triazole (3AT) was added to inhibit the leaky HIS3 expression. (E) Rad53 phosphorylates Mck1 in vitro. Mck1-5FLAG was precipitated from yeast cells and incubated with purified recombinant His6-RAD53 and His6-rad53-KD (K227A) in the presence of ɣ-³²P-ATP as detailed in Methods. After resolved in an 8% polyacrylamide gel with SDS, the samples were subjected to autoradiography. Then, the gel was stained by Coomassie Brilliant Blue (CBB) to show the amount of the loaded protein in each reaction.