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In response to genomic insults cells trigger a signal transduction pathway, known as DNA damage checkpoint, whose role is to help the cell to cope with the damage by coordinating cell cycle progression, DNA replication and DNA repair mechanisms. Accumulating evidence suggests that activation of the first checkpoint kinase in the cascade is not due to the lesion itself, but it requires recognition and initial processing of the lesion by a specific repair mechanism. Repair enzymes likely convert a variety of physically and chemically different lesions to a unique common structure, a ssDNA region, which is the checkpoint triggering signal. Checkpoint kinases can modify the activity of repair mechanisms, allowing for efficient repair, on one side, and modulating the generation of the ssDNA signal, on the other. This strategy may be important to allow the most effective repair and a prompt recovery from the damage condition. Interestingly, at least in some cases, if the damage level is low enough the cell can deal with the lesions and it does not need to activate the checkpoint response. On the other hand if damage level is high or if the lesions are not rapidly repairable, checkpoint mechanisms become important for cell survival and preservation of genome integrity.

Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G(1) phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Delta dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.

DNA interstrand cross-links (ICLs) are highly cytotoxic DNA lesions hindering DNA replication and transcription. Whereas in bacteria and yeast the molecular mechanisms involved in ICL repair are genetically well dissected, the scenario in multicellular organisms remains unclear. Here, we report that the two new mus308 genes, polq-1 and hel-308 are involved in ICL repair in Caenorhabditis elegans. After treatment with ICL agents, a decrease in survival and an increase in checkpoint-induced cell-cycle arrest and apoptosis of germ cells is observed in mutants of both genes. Although sensitive to ICL agents and to a minor extent to IR, cytological and epistatic analyses suggest that polq-1 and hel-308 are involved in different DNA repair pathways. While hel-308 functions in a Fanconi anemia-dependent pathway, polq-1 has a role in a novel distinct and brc-1 (CeBRCA1)-dependent ICL repair process in metazoans.

Translesion DNA synthesis (TLS) is one of the mechanisms involved in lesion bypass during DNA replication. Three TLS polymerases (Pol) are present in the yeast Saccharomyces cerevisiae: Pol zeta, Pol eta and the product of the REV1 gene. Rev1 is considered a deoxycytidyl transferase because it almost exclusively inserts a C residue in front of the lesion. Even though REV1 is required for most of the UV-induced and spontaneous mutagenesis events, the role of Rev1 is poorly understood since its polymerase activity is often dispensable. Rev1 interacts with several TLS polymerases in mammalian cells and may act as a platform in the switching mechanism required to substitute a replicative polymerase with a TLS polymerase at the sites of DNA lesions. Here we show that yeast Rev1 is a phosphoprotein, and the level of this modification is cell cycle regulated under normal growing conditions. Rev1 is unphosphorylated in G1, starts to be modified while cells are passing S phase and it becomes hyper-phosphorylated in mitosis. Rev1 is also hyper-phosphorylated in response to a variety of DNA damaging agents, including treatment with a radiomimetic drug mostly causing double-strand breaks (DSB). By using the chromosome spreading technique we found the Rev1 is bound to chromosomes throughout the cell cycle, and its binding does not significantly increase in response to genotoxic stress. Therefore, Rev1 phosphorylation does not appear to modulate its binding to chromosomes, suggesting that such modification may influence other aspects of the TLS process. Rev1 binding under damaged and undamaged conditions, is at least partially dependent on MEC1, a gene playing a pivotal role in the DNA damage checkpoint cascade. This genetic dependency may suggest a role for MEC1 in spontaneous mutagenesis events, which require a functional REV1 gene.

Eukaryotic cells respond to a variety of DNA insults by triggering a common signal transduction cascade, known as checkpoint response, which temporarily halts cell-cycle progression. Although the main players involved in the cascade have been identified, there is still uncertainty about the nature of the structures that activate these surveillance mechanisms. To understand the role of nucleotide excision repair (NER) in checkpoint activation, we analyzed the UV-induced phosphorylation of the key checkpoint proteins Chk1 and p53, in primary fibroblasts from patients with xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy (TTD), or UV light-sensitive syndrome. These disorders are due to defects in transcription-coupled NER (TC-NER) and/or global genome NER (GG-NER), the NER subpathways repairing the transcribed strand of active genes or the rest of the genome, respectively. We show here that in G0/G1 and G2/M phases of the cell cycle, triggering of the DNA damage cascade requires recognition and processing of the lesions by the GG-NER. Loss of TC-NER does not affect checkpoint activation. Mutations in XPD, XPB, and in TTDA, encoding subunits of the TFIIH complex, involved in both transcription and NER, impair checkpoint triggering. The only exception is represented by mutations in XPD, resulting in combined features of XP and CS (XP/CS) that lead to activation of the checkpoint cascade after UV radiation. Inhibition of RNA polymerase II transcription significantly reduces the phosphorylation of key checkpoint factors in XP/CS fibroblasts on exposure to UV damage.

The use of translesion synthesis (TLS) polymerases to bypass DNA lesions during replication constitutes an important mechanism to restart blocked/stalled DNA replication forks. Because TLS polymerases generally have low fidelity on undamaged DNA, the cell must regulate the interaction of TLS polymerases with damaged versus undamaged DNA to maintain genome integrity. The Saccharomyces cerevisiae checkpoint proteins Ddc1, Rad17, and Mec3 form a clamp-like structure (the 9-1-1 clamp) that has physical similarity to the homotrimeric sliding clamp proliferating cell nuclear antigen, which interacts with and promotes the processivity of the replicative DNA polymerases. In this work, we demonstrate both an in vivo and in vitro physical interaction between the Mec3 and Ddc1 subunits of the 9-1-1 clamp and the Rev7 subunit of the Polzeta TLS polymerase. In addition, we demonstrate that loss of Mec3, Ddc1, or Rad17 results in a decrease in Polzeta-dependent spontaneous mutagenesis. These results suggest that, in addition to its checkpoint signaling role, the 9-1-1 clamp may physically regulate Polzeta-dependent mutagenesis by controlling the access of Polzeta to damaged DNA.

The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process. Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss of H3-Lys(79) methylation does not affect Mec1 activation, whereas it renders cells checkpoint-defective by preventing phosphorylation of Rad9. Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase.

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.

Rad17-Mec3-Ddc1 forms a proliferating cell nuclear antigen-like complex that is required for the DNA damage response in Saccharomyces cerevisiae and acts at an early step of the signal transduction cascade activated by DNA lesions. We used the mec3-dn allele, which causes a dominant negative checkpoint defect in G1 but not in G2, to test the stability of the complex in vivo and to correlate its assembly and disassembly with the mechanisms controlling checkpoint activation. Under physiological conditions, the mutant complex is formed both in G1 and G2, although the mutant phenotype is detectable only in G1, suggesting that is not the presence of the mutant complex per se to cause a checkpoint defect. Our data indicate that the Rad17-Mec3-Ddc1 complex is very stable, and it takes several hours to replace Mec3 with Mec3-dn within a wild type complex. On the other hand, the mutant complex is rapidly assembled when starting from a condition where the complex is not pre-assembled, indicating that the critical factor for the substitution is the disassembly step rather than complex formation. Moreover, the kinetics of mutant complex assembly, starting from conditions in which the wild type form is present, parallels the kinetics of checkpoint inactivation, suggesting that the complex acts in a stoichiometric way, rather than catalytically.

DNA polymerase alpha (pol alpha) holds a special position among the growing family of eukaryotic DNA polymerases. In fact, pol alpha is associated with DNA primase to form a four subunit complex and, as a consequence, is the only enzyme able to start DNA synthesis de novo. Because of this peculiarity the major role of the DNA polymerase alpha-primase complex (pol-prim) is in the initiation of DNA replication at chromosomal origins and in the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. However, pol-prim seems to play additional roles in other complex cellular processes, such as the response to DNA damage, telomere maintenance, and the epigenetic control of higher order chromatin assembly.

Eukaryotic cells must be able to coordinate DNA repair, replication and cell cycle progression in response to DNA damage. A failure to activate the checkpoints which delay the cell cycle in response to internal and external cues and to repair the DNA lesions results in an increase in genetic instability and cancer predisposition. The use of the yeast Saccharomyces cerevisiae has been invaluable in isolating many of the genes required for the DNA damage response, although the molecular mechanisms which couple this regulatory pathway to different DNA transactions are still largely unknown. In analogy with prokaryotes, we propose that DNA strand breaks, caused by genotoxic agents or by replication-related lesions, trigger a replication coupled repair mechanism, dependent upon recombination, which is induced by the checkpoint acting during S-phase.

The highly conserved DNA polymerase alpha-primase complex (pol-prism) is the only eukaryotic DNA polymerase that can initiate DNA synthesis de novo. It is required both for the initiation of DNA replication at chromosomal origins and for the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. The dual role of pol-prim makes it a likely target for mechanisms that control cell-cycle S-phase entry and progression.

The in vivo function of the 34 kDa subunit of yeast replication protein A (RPA), encoded by the RFA2 gene, has been studied by analyzing the effect of Rpa34 depletion and by producing and characterizing rfa2 temperature-sensitive mutants. We show that unbalanced stoichiometry of the RPA subunits does not affect cell growth and cell cycle progression until the level of Rpa34 becomes rate-limiting, at which point cells arrest with a late S/G2 DNA content. Rpa34 is involved in DNA replication in vivo, since rfa2 ts mutants are defective in S phase progression and ARS plasmid stability, and rfa2 pol1 double mutants are non-viable. Moreover, when shifted to the restrictive temperature, about 50% of the rfa2 mutant cells rapidly die while traversing the S phase and the surviving cells arrest in late S/G2 at the RAD9 checkpoint. Finally, rfa2 mutant cells have a mutator and hyper-recombination phenotype and are more sensitive to hydroxyurea and methyl-methane-sulfonate than wild-type cells.

The POL1 gene, encoding DNA polymerase alpha (pol alpha) in Saccharomyces cerevisiae, is transiently transcribed during the cell cycle at the G1/S phase boundary. Here we show that yeast pol alpha is present at every stage of the cell cycle, and its level only slightly increases following the peak of POL1 transcription. POL1 mRNA synthesis driven by a GAL1 promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pol alpha polypeptide drops below the physiological level. Moreover, alpha-factor-arrested cells can enter S phase and divide synchronously even if POL1 transcription is abolished. These results indicate that the level of yeast pol alpha is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase.

We report the 9210 bp sequence from a segment of yeast chromosome III cloned from strain AB972 in lambda PM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24-24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24-24A and cause a quite significant rearrangement of the whole region. We performed functional analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24-24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain-dependent effect on sensitivity to amino acid balance and to 3-aminotriazole, when cells were grown in synthetic medium.

Eukaryotic DNA primases are composed of two distinct subunits of 48-50 and 58-60 kDa. The amino acid sequences derived from the nucleotide sequences of the cloned genes are known only for the yeast and mouse polypeptides, and the extensive homology between the corresponding mouse and yeast subunits suggests conservation of functional domains. We were able to express in Saccharomyces cerevisiae the homologous and mouse primase-encoding genes under the control of both the constitutive ADH1 and the inducible GAL1 strong promoters, thus obtaining strains producing relevant amounts of the different polypeptides. In vivo complementation studies showed that neither one of the wild-type mouse primase-encoding genes was able to rescue the lethal or temperature-sensitive phenotype caused by mutations in the yeast PRI1 or PRI2 genes, indicating that these proteins, even if structurally and functionally very similar, might be involved in critical species-specific interactions during DNA replication.

We have analyzed the effects of temperature-sensitivity (ts)-conferring mutations in the Saccharomyces cerevisiae DNA polymerase I-encoding gene on cell growth, in vivo DNA synthesis, intrachromosomal gene conversion and pop-out recombination. Also, we have identified the molecular defect responsible for the ts phenotype. Two mutant alleles (cdc17-1, cdc17-2) were originally identified as cell-cycle mutations, while a third mutation (hpr3) was found during a genetic screening for mutants with a hyper-recombination phenotype. Both cdc17-2 and hpr3 cells complete one round of cell division and DNA replication after shift to nonpermissive temperature, before being arrested as dumbbell-shaped cells. Conversely, the cdc17-1 mutation immediately blocks growth and DNA synthesis at 37 degrees C. No substantial difference was observed in the frequency of intrachromosomal gene conversion and pop-out recombination events, when hpr3 and cdc17-1 were compared to the previously characterized pol1-1 mutant. These two frequencies were ten- to 30-fold above wild-type level at semipermissive temperature. In each mutant, a single bp substitution, causing the replacement of Gly residues by either Asp (cdc17-1, cdc17-2) or Glu (hpr3) in yeast DNA polymerase I is responsible for the ts phenotype.

Partially purified cell-free extracts prepared from growing cells of the yeast Saccharomyces cerevisiae catalyze DNA synthesis directed by the chimeric plasmid BTYP-2 containing the whole 2 μm DNA sequence. The biochemical properties of the reaction have been examined and inactivation studies indicate that DNA synthesis is only due to protein factors. Analysis of the products of the DNA synthesis reaction demonstrates that part of the in vitro synthesized DNA is not covalently linked to the template, thus suggesting initiation of new DNA chains. The analysis of the distribution of the labelled products at early times of reaction showed a preferential synthesis on the 2 μm portion of the BTYP-2 DNA template.

The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2. Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy. Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest. Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.

Terminal Transferase (TdT), Adenosine Deaminase (ADA), immunological membrane markers, cytochemical reactivity and cytogenetics were analyzed in 226 patients with ALL, AUL and AML, in 70 patients with CML and in 3 cases of Ph' positive acute leukemia presenting as ALL. TdT was tested in peripheral blood and bone marrow with both the biochemical and immunofluorescence (IF) methods, and ADA was determined biochemically only in peripheral blood cells. By using conventional cytochemistry, cell surface markers determinations, TdT and ADA analysis, three distinct groups are recognized in ALL at presentation: T-ALL with TdT+ and very high ADA values; non-T, non-B ALL with TdT+ and intermediate levels of ADA; B-ALL with TdT absence and low levels of ADA. Clinical presentation and responses to therapy in adult and children ALL were correlated to TdT determinations. The median survivals in adults, calculated for TdT+ and TdT- groups, were 14.2 and 5.6 months, respectively. TdT and ADA were determined in ALL during remission. The wide fluctuation observed for TdT IF and ADA values prevented a reliable monitoring of remissions. At relapse, TdT and ADA values were similar to those found for ALL at presentation; TdT IF determinations were diagnostic in cases showing CNS involvement as the only localization. Forty per cent of AUL and 11% of AML cases were positive for TdT; the medians of ADA values of the TdT+ cases in both AML and AUL were several times higher than those obtained in the TdT- group. While TdT positivity and high ADA had a favorable prognostic value in AUL, similar conclusions can not be drawn at the moment for AML. In chronic phase of CML, TdT was strictly negative and ADA values were increased over the control line only in cases showing initial signs of transformation. In acute phase, the cases positive for TdT (32%) presented a significantly higher ADA activity than the TdT negative ones. The actuarial survival curves for the TdT+ and TdT- groups differ significantly, presenting median survivals from onset of phase of 11 and 4.8 months respectively. The three cases of Ph' positive ALL were all TdT+, presented high ADA values and entered chronic phase of CML after therapy.

Partially overlapping fragments of the gene encoding yeast DNA polymerase I have been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector lambda gt11. The three gene fragments we analyzed in detail encode part of a yeast protein that has been identified as yeast DNA polymerase I, because it shares with this enzyme a number of antigenic determinants. In fact, the yeast protein fragments expressed by the recombinant phages react with both polyclonal and monoclonal antibodies raised against different, highly purified preparations of DNA polymerase I. Moreover, they can be used to affinity purify antibodies specifically reacting with active DNA polymerase I polypeptides and they compete with the yeast enzyme for binding to antibodies that inhibit catalytic activity. The gene is located on chromosome XIV in the yeast genome, and it is transcribed as a 5.2 kb mRNA.

The PRI1 gene of Saccharomyces cerevisiae encodes for the p48 polypeptide of DNA primase. We have determined the nucleotide sequence of a 1,965 bp DNA fragment containing the PRI1 locus. The entire coding sequence of the gene lies within an open reading frame, and there are 409 amino acids in the single polypeptide protein if translation is assumed to start at the first ATG in this frame. The 5' and 3' end-points of PRI1 mRNA have been determined by S1 mapping and primer extension analysis. The primary structure and the codon usage of PRI1 suggest that this essential gene is poorly expressed in yeast cells.

Bacillus subtilis strain PB 2427 temperature sensitive in the synthesis of RNA during spore germination and outgrowth has been characterized to some extent. At non permissive temperature (46 degrees C) strain PB 2427 synthesizes stable and unstable RNA for 50 min from the beginning of germination and then stops. Most of the stable RNA is degraded to shorter molecules but can be identified as ribosomal RNA by hybridization-competition experiments. At non permissive temperature, in the presence of chloramphenicol, synthesis of RNA proceeds, though at a reduced rate, for at least 90 min. By hybridization-competition experiments it can also be shown that the RNA synthesized at 46 degrees C in the presence of chloramphenicol includes transcripts that are absent, from the RNA synthesized at 46 degrees C in the absence of drug. The RNA polymerase (holo and core) purified from vegatative cells of the mutant strain does not appear to have a greater heat-lability as compared with the enzyme purified from the parental strain. At non permissive temperature only six polypeptide chains with MW ranging from 47,000 to 78,000 daltons are synthesized by the germinating spores of the mutant.

A procedure to prepare crude extracts from single colonies of Saccharomyces cerevisiae is described. Partially purified extracts catalyzed DNA synthesis directed by single-stranded fd DNA. Maximum activity requires the presence of ribonucleoside triphosphates although extensive DNA synthesis is observed in the presence of only deoxynucleoside triphosphates. Alkaline sucrose gradient analysis demonstrated that initiation of new DNA chains occurs in vitro and the DNA products synthesized are heterogeneous in size, Isopycnic analysis of the products of ribonucleotide-initiated fd DNA replication showed covalent linkage between the initiator RNA and the newly synthesized DNA. The fd-replicating capacity of extracts prepared from cell-division-cycle mutants, defective in events controlling DNA initiation of elongation, showed an increased thermosensitivity in the DNA replication reaction in vitro.

Two mutants of Bacillus subtilis temperature-sensitive in RNA synthesis were isolated. One mutation (rna-20) was demonstrated to be an allele of a previously identified gene (Riva et al., 1976). The other mutation (rna-16) identified a different gene and was mapped near aroI. The rna-16 mutation at the permissive temperature affected the spore outgrowth process. Purified RNA polymerase from rna-16 did not show any temperature sensitivity or structural defect.

The immunopurified yeast DNA-polymerase-I--DNA-primase complex synthesizes oligo(rA) and oligo(rG) molecules that are used as primer for replication of poly(dT) and poly(dC). Neither initiation nor DNA synthesis is observed with poly(dA) and poly(dI). Nitrocellulose-filter binding shows that the enzyme complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Although the yeast complex initiates DNA synthesis on deoxypyrimidine homopolymers, it prefers to elongate pre-existing primer molecules rather than to initiate de novo DNA replication. The size of the oligo(rA) and oligo(rG) primer molecules has been determined by urea/polyacrylamide gel electrophoresis: longer oligoribonucleotides are synthesized when their utilization is prevented by omitting dNTP. An oligodeoxythymidylate template with a chain length as short as five residues can support oligo(rA) synthesis catalyzed by the yeast DNA-polymerase--DNA-primase complex and the size of the oligoribonucleotide products synthesized with oligodeoxythymidylate of differing chain length has also been determined. The mechanistic properties of the DNA-polymerase--DNA-primase complexes, purified from different eukaryotic organisms, appear to be very similar. The possible biological implication of the studies on the mechanism and specificity of initiation of DNA synthesis in a well-defined model template system has been discussed.