The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.

RAD9, RAD17, and RAD24 are required for S phase regulation in Saccharomyces cerevisiae in response to DNA damage.

We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9 delta, rad 17 delta, and rad24 delta. Furthermore, the triple rad9 delta rad17 delta rad24 delta mutant also has a less severe defect than mec1-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1 delta, mag1 delta, apn1 delta, rev3 delta, rad18 delta, rad16 delta, dun1-delta 100, sad4-1, tel1 delta, rad26 delta, rad51 delta, rad52-1, rad54 delta, rad14 delta, rad1 delta, pol30-46, pol30-52, mad3 delta, pds1 delta/esp2 delta, pms1 delta, mlh1 delta, and msh2 delta are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation.

Feedback inhibition of the yeast ribosomal protein gene CRY2 is mediated by the nucleotide sequence and secondary structure of CRY2 pre-mRNA.

The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells. Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA. Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1. cis-Acting elements involved in repression of CRY2 were identified by assaying the expression of CRY2-lacZ gene fusions and promoter fusions in CRY1 CRY2 and cry1-delta CRY2 strains. Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron. Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression. The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis. Wild-type cells contain very low levels of both CRY2 pre-mRNA and CRY2 mRNA. Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm. Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally. A model for coupling ribosome assembly and regulation of ribosomal protein gene expression is proposed.

A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage.

We demonstrate that in S. cerevisiae the rate of ongoing S phase is slowed when the DNA is subjected to alkylation. Slowing of replication is dependent on the MEC1 and RAD53 genes, indicating that lesions alone do not slow replication in vivo and that the slowing is an active process. While it has been shown that a MEC1- and RAD53-dependent checkpoint responds to blocked replication or DNA damage by inhibiting the onset of mitosis, we demonstrate that this checkpoint must also have an additional target within S phase that controls replication rate. MEC1 is a homolog of the human ATM gene, which is mutated in ataxia telangiectasia (AT) patients. Like mec1 yeast, AT cells are characterized by damage-resistant DNA synthesis, highlighting the congruence of the yeast and mammalian systems.

Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family.

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype. (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.

Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits.

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

Pancreatic tumor pathogenesis reflects the causative genetic lesion.

Transgenic mice in which c-myc expression is targeted to pancreatic acinar cells develop mixed acinar/ductal pancreatic adenocarcinomas between 2 and 7 months of age. This contrasts with the effect on pancreas of the simian virus 40 tumor antigen or activated ras, which in adult mice causes lesions composed exclusively of acinar-like cells. Furthermore, during an early stage of myc-induced pathology, transformed acinar-derived cells appear within islets, suggesting that islet hormones may influence the progression of these exocrine pancreatic tumors. These findings demonstrate that the initial oncogenic alteration can influence the pattern of subsequent tumor pathogenesis and, given that human exocrine pancreatic tumors are predominantly ductal adenocarcinomas, support the suggestion that transformed acinar cells may contribute to the genesis of this serious disease in man.

Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly.

Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribosomal RNAs. However, most ribosomal proteins and ribosomal RNAs that are components of the same subunit as L16 or rp59 are rapidly degraded upon depletion of L16 or rp59, presumably resulting from abortive assembly of the subunit. Depletion of L16 has no effect on the stability of most components of the 40S subunit. Conversely, termination of synthesis of rp59 has no effect on the stability of most 60S subunit components. The implications of these findings for control of ribosome assembly and the order of assembly of ribosomal proteins into the ribosome are discussed.