FIG. 8.

H2A-AQE cells show defects in HU-induced cell cycle arrest in the absence of S-phase checkpoint proteins. (A) H2A-AQE mutations make cells with mutations in the S-phase checkpoint (mrc1Δ and cds1Δ), but not those with mutations in the DNA damage checkpoint (crb2Δ and chk1Δ), more sensitive to HU. Viability of cells with indicated genotypes was measured by counting colony formation on YES plates after treatment with 12 mM HU for the indicated lengths of time. Strains used were wild type (TMN2665), hta1 hta2 (TMN3293), crb2Δ (TMN2941), crb2Δ hta1 hta2 (TMN3297), chk1Δ (TMN2943), chk1Δ hta1 hta2 (TMN3298), mrc1Δ (TMN3326), mrc1Δ hta1 hta2 (TMN3327), cds1Δ (TMN2945), cds1Δ hta1 hta2 (TMN3324), mrc1Δ chk1Δ (TMN3328), and cds1Δ chk1Δ (TMN3325). (B) Cells of the indicated genotypes (the same strains as in panel A) were mock treated or treated with 12 mM HU for 6 h and then stained with DAPI. Increased occurrence of the cut phenotype in mrc1Δ hta1-S129A hta2-S128A and cds1Δ hta1-S129A hta2-S128A cells, but not in chk1Δ hta1-S129A hta2-S128A cells, is consistent with a defect in DNA damage checkpoint enforced by Chk1. (C) Fivefold serial dilutions of mrc1Δ and mrc1Δ hta1-S129A hta2-S128A cells carrying either the control pAL-SK+ plasmid or the Cds1 overexpression plasmid pAL-Cds1 (TMN3329, TMN3330, TMN3331, and TMN3332) were plated on minimal medium with the indicated concentrations of HU. Pictures were taken after 6 days at 32°C. (D) Fivefold serial dilutions of cells with the indicated genotypes (the same strains as used in panel A) were plated onto YES with the indicated concentrations of CPT. Pictures were taken after 4 days at 32°C.