(A and B) Genetic interactions of chl1::HIS3 with mutations in DNA replication genes. Cells were streaked on YEPD plates, which were incubated at indicated temperatures for 2 days. The strains used were wild-type (wt), AP22; chl1, AP22Dchl1; cdc6-3, YB0297; orc5-1, JRY4245; mcm2-1, M46-3C; Double mutants were constructed by disrupting/deleting CHL1, CHL4 or MCM22 in these strains as described in Supplementary Data.

The chl1 mutation confers growth sensitivity in the presence of MMS and UV rays. (A) Spot assay for MMS sensitivity of chl1::HIS3 (AP22Dchl1), AP22Δ22 (mcm22-Δ1::TRP1), and wild-type (AP22) strains. Growing cells were serially diluted and spotted on YEPD plates containing 0.025% MMS or no MMS (control). The YEPD plate was incubated at 30°C for 2 days while the MMS-containing plate was incubated at the same temperature for 3–4 days. (B) chl1 mutation confers hypersensitivity to UV rays. Exponentially growing cells, appropriately diluted, were plated on YEPD plates, exposed to UV rays for various times and incubated in the dark at 30°C. Control plates were also kept which were not irradiated and were used to calculate total number of cells plated. Colonies were counted after 2–3 days of incubation. Results are the mean values of four independent experiments and error bars are SD values from the mean.

Chl1p is required for DNA damage repair in S-phase. (A) A schematic representation of the mechanism by which alkylated DNA can give rise to nicks, gaps and double-strand breaks, resulting in higher mobility of genomic DNA during agarose gel electrophoresis. Alkylated bases are cleaved by DNA glycosylases creating apurinic or apyrimidinic (AP) sites which are cleaved by AP endonuclease/lyase giving rise to single-strand breaks. Closely opposed and unrepaired single-strand breaks (SSB), coupled with the stalling of replication forks at alkylated bases can give rise to double-strand breaks in DNA (59) which are repaired in the wild-type but not in the mutant. (B and C) Overnight cultures of wild-type (SL14) and mutant strains chl1 (SL14Dchl1) grown at 30°C in liquid YEPD were re-inoculated in YEPD at 30°C till mid-log-phase (OD₆₁₀ ≅ 0.8–1) and synchronized in G1 using alpha-factor. Cells were released from arrest in liquid YEPD at 30°C in the presence or absence of varying concentrations of MMS. At indicated times, aliquots were taken out for FACS analysis (C) and to isolate DNA in agarose plugs as described in Methods section. Agarose gel (0.5%) electrophoresis of DNA was carried out for 28 h at 2.5 V/cm, as described under Methods (B). (D) Overnight cultures of wild-type (SL14) and mutant strains chl1 (SL14Dchl1) and mec1-1 (US3138), grown at 23°C in liquid YEPD were re-inoculated in YEPD at 28°C till mid-log phase (OD₆₁₀ ≅ 0.8–1) and synchronized in G1 using alpha-factor. Cells were released from arrest in liquid YEPD at 28°C in the presence or absence of 0.035% MMS. At indicated times, aliquots were taken out for FACS analysis (D) and to isolate DNA in agarose plugs as described in Methods. (E) Agarose gel electrophoresis of the DNA in plugs from (D) was carried out as described above. There were no differences in the mobilities of genomic DNA isolated from cultures not exposed to MMS at all time points in the experiment. DNA from untreated cells (−MMS) corresponds to genomic DNA isolated 1.5 h after release from arrest of untreated cells, that is, 1 h after MMS wash. ‘−’ indicates no MMS was added while ‘+’ indicates MMS added. (F) Growth of wild-type SL14 (CHL1 MEC1), SL14Dchl1 (chl1 MEC1) and US3138 (mec1-1) on YEPD plates with or without 0.01% MMS. Plates were incubated for 3 days at 23°C. (G) Agarose gel electrophoresis (as described above) of DNA isolated in plugs from wild-type (A3), chl1 (A3Dchl1), mcm2-1 (SL13) and mcm2-1 chl1 (SL13Dchl1) cells grown in liquid YEPD at 32°C for 24 h.

Cell viability, G2/M arrest and nuclear fragmentation in wild-type, chl1, mcm2-1 and mcm2-1 chl1 cells. A3 (MCM2 CHL1), A3Dchl1 (MCM2 chl1), SL13 (mcm2-1 CHL1) and SL13Dchl1 (mcm2-1 chl1) cells were grown in YEPD at 23°C to log-phase and re-inoculated at 32°C in pre-warmed YEPD medium. Aliquots were removed at various times for determining cell viabilities (A), for fluorescence activated cell sorting (FACS) analysis (B) and stained with DAPI for nuclear morphology (C). Samples from a few representative time points are shown for (B) and (C) h, hours. (D and E) The same strains were arrested in G1 using alpha-factor and released at 32°C in fresh YEPD. Aliquots were removed at time intervals for monitoring S-phase progression using flow cytometry and for budding index (fraction of cells that had initiated budding after release from G1 arrest). (F) Cell viabilities of wild-type, chl1, orc5-1, orc5-1 chl1, cdc6-3 and cdc6-3 chl1 grown at 32°C. 699 (wild-type), 699Dchl1 (chl1), JRY4245 (orc5-1), JRY4245Dchl1 (orc5-1 chl1), YB0297 (cdc6-3) and YB0297Dchl1 (cdc6-3 chl1) were grown to exponential phase in liquid YEPD at 23°C, re-inoculated into pre-warmed liquid YEPD at 32°C and processed for the determination of cell viabilities as in (A). (G) Nuclear fragmentation in orc5-1 chl1 and cdc6-3 chl1 cells. JRY4245 (orc5-1), JRY4245Dchl1 (orc5-1 chl1), YB0297 (cdc6-3) and YB0297Dchl1 (cdc6-3 chl1) were grown to exponential phase in liquid YEPD at 23°C, re-inoculated into pre-warmed liquid YEPD at 32°C and grown for 4.5 h. Thereafter, cells were stained with DAPI and analyzed for their morphologies using the fluorescence microscope. Abbreviations: h, hours; min, minutes; exp, exponential.

DNA damage checkpoint is active in chl1 mutant cells. (A) S-phase progression of mutant and wild-type cells in the presence of MMS. 699 (CHL1 RAD24 RAD53), 699Dchl1 (chl1), SL3 (rad24), SL4 (rad24 chl1) and SL7 (rad53-21) cells were all synchronized with alpha-factor, washed free of alpha-factor, resuspended in pre-warmed YEPD medium at 30°C, divided into two parts and MMS was added to a final concentration of 0.035% to one part. Both the cultures were kept shaking at 30°C. Aliquots were removed at various times for FACS analysis. Arrows indicate G1 and G2 DNA contents. (B) chl1 are proficient in Rad53p phosphorylation in response to MMS treatment in S-phase. SL14 (CHL1 RAD24), SL14Dchl1 (chl1), SS1 (rad24) and SS1Dchl1 (rad24 chl1) were arrested in G1 phase and released in fresh YEPD medium containing 0.035% MMS at 30°C. Rad53p phosphorylation was detected by western blot analysis of proteins extracted from aliquots of cells removed at indicated times, using antibodies directed against the Rad53 protein. (C) DNA content of cells from the same aliquots analyzed by flow cytometry. (D) chl1 cells are hypersensitive towards killing by MMS in S-phase. 699 (wild-type), 699Dchl1 (chl1), SL3 (rad24), SL3Dchl1 (rad24 chl1), 699Δsgs1 (sgs1), 699Δsgs1Dchl1 (sgs1 chl1), SL7 (rad53-21), SL7Δchl1 (rad53-21 chl1) cells were arrested by alpha-factor in G1 and released in fresh YEPD containing 0.035% MMS. Aliquots were removed for cell viabilities and for DNA content. Data are averages of two independent experiments which gave very similar results. Bars are deviations from the average values and are not shown in rad24 and rad24 chl1 series for the sake of clarity in rad53 and rad53 chl1 series. (E) DNA content of cells in (D) measured by flow cytometry. Arrows indicate G1 and G2 DNA contents. Abbreviations are as in Figure 2.