lsm1Δ cells are competent to repair DSBs by HR and NHEJ. (A) Sensitivity of different mutants to DNA-damaging drugs. (B) Colonies formed by different strains in YPD medium after 2 days of growth at 30°C. (C) Schematic representation of the strain GA2321 used to monitor HR-dependent repair of HO endonuclease-induced DSB. In this strain, both HML and HMR loci are deleted and a non-cleavable copy of the MAT locus is integrated on chromosome V. Expression of HO endonuclease is controlled by the GAL1,10 promoter. Cells were grown overnight in a raffinose-containing medium and then plated on media containing galactose (HO ON) or glucose (HO OFF). (D) Plasmid map of the vector pBTM116 used to monitor NHEJ repair. (E) Percentage of transformant recovery. Each strain was transformed with equal amounts of linear or uncut plasmid, and the number of colonies obtained with the linearized plasmid was normalized to the obtained from uncut plasmid.

Lsm1 is required for the recovery of replication-fork stalling induced by nucleotide depletion (HU) and at natural pause sites. (A) Prolonged Rad53 checkpoint kinase activation upon removal of HU in lsm1Δ cells. The phosphorylation state of Rad53 was determined by western blot at the indicated time points. (B) Cell-cycle progression after treatment with HU is delayed in lsm1Δ cells. DNA contents of WT and lsm1Δ cells were determined by FACS analysis of mid-log phase cultures (ASN), cells synchronized in S-phase after treatment with 200 mM HU and at the indicated times after removal of the drug. (C) lsm1Δ cells accumulate ERCs. ERCs were detected by Southern blot hybridization using a radioactive rDNA probe. A shorter exposure of the signal corresponding to the rDNA array is shown in the upper panel. (D) Synthetic fitness interaction between lsm1Δ, mms4Δ and mus81Δ mutants.

lsm1Δ cells accumulate free histones that make replication forks sensitive to drug-induced replication-fork stalling. (A) Degradation of overexpressed histones is impaired in lsm1Δ cells. Wild-type and lsm1Δ strains carrying the 2 μ-URA3-GAL-HA-HHT2 plasmid (pGAL-H3) were grown overnight in minimal medium lacking uracil and with raffinose as a carbon source. Cells were synchronized in G1 with α factor for 2 h and galactose was added to the medium for 1 h. Half of the cultures were then collected and transferred to a glucose-containing medium, and the other half was transferred to the same medium containing galactose, but with the addition of cycloheximide at 35 μg/ml. Samples were taken every 30 min after switching to glucose or cycloheximide. (B) Effect of histone overexpression on the growth of WT, lsm1Δ and lsm1Δ ski2Δ strains. Strains transformed with empty vector or with plasmids expressing histones H3, H4 or H2A–H2B under the control of GAL promoter were grown overnight in minimal medium without uracil and with 2% raffinose as a carbon source. Five-fold serial dilutions of each strain were plated on the same medium, either with glucose or galactose, as carbon source. The plates were incubated for 4 days at 30°C. (C) Deletion of either hht2-hhf2 or hta1htb1 histone gene pairs significantly suppresses the DNA damage sensitivity of the lsm1Δ and lsm1Δ ski2Δ strains.

lsm1Δ cells are hypersensitive to DNA damage, proficient to activate the DNA damage checkpoint and accumulate DNA DSBs. (A) Sensitivity of wild-type and lsm1Δ cells to different drugs. (B) Phosphorylation of Rad53 in response to DNA damage. Cells were treated with 0.033% MMS, 2 μg/ml Phl or 0.2 M HU for the indicated time periods. (C) Percentage of cells containing Rad52-YFP foci in wild-type and lsm1Δ strains untreated or treated with 0.02% MMS for 2 and 4 h.

Lsm1 is necessary for the recovery of replication-fork stalling induced by MMS. (A) Schematic representation of the experiment and FACS analysis of the S-phase progression in wild-type and lsm1Δ cells in the presence of 0.033% MMS. (B) Percentage of survival after treatment with 0.033% MMS. (C) Schematic representation of the experiment and Rad53 checkpoint kinase activation and histone H2A phosphorylation upon recovery from MMS damage in wild-type and lsm1Δ cells. (D) Analysis of completion of DNA replication by PFGE. Cells were treated as in (C) and samples were collected at the indicated points, embedded in agarose plugs and analysed by PFGE.

Sensitivity to DNA-damaging drugs and histone mRNA levels in mRNA decay mutants. (A) Deletion of SKI2 increases sensitivity of lsm1Δ cells to DNA damage. Five-fold serial dilution of cultures of the indicated mutants were spotted on YPD or YPD containing different drugs at the indicated concentrations. (B) Lsm1 controls histone mRNA levels. Total RNA from the indicated strains were separated on a agarose formaldehyde gel and sequentially hybridized with different histone probes. Histone mRNAs was quantitated by densitometry analysis using Image J. The amount of mRNA was plotted against time to determine the half-lives of histone mRNAs. (C) Histone H3 mRNA levels after treatment with 0.2 M HU. Total rRNA stained with methylene blue was used as loading controls.