Involvement In DNA Replication Research Articles

Targeting tumour-supportive cellular machineries in anticancer drug development

Traditional anticancer chemotherapeutics targeting DNA replication and cell division have severe side effects, but they have proved to be highly successful in treating some cancers. Drugs targeting signalling oncoproteins that have gained tumour-driving functions through mutations or overexpression were subsequently developed to increase specificity and thus reduce side effects, but have limitations such as the development of resistance. Now, a new wave of small-molecule anticancer agents is emerging, targeting complex multicomponent cellular machineries - including chromatin modifiers, heat shock protein chaperones and the proteasome - which thus interfere with those support systems that are more essential for cancer cells than for normal cells. Here, we provide our perspective on the advantages and limitations of agents that target tumour-supportive cellular machineries (other than those involving DNA replication), comparing them with agents that target signalling intermediates.

Functional and structural analysis of the major amidase (Atl) in Staphylococcus

The cytoplasmic membrane of most bacteria is surrounded by a more or less thick murein layer (peptidoglycan) that protects the protoplast from mechanical damage, osmotic rupture and lysis. When bacteria are dividing processes are initiated stepwise that involve DNA replication, constriction of the membranes, cell growth, biosynthesis of new murein, and finally the generation of two daughter cells. As the daughter cells are still covalently interlinked by the murein network they must be separated by specific peptidoglycan hydrolases, also referred to as autolysins. In staphylococci, the major autolysin (Atl) and its processed products N-acetylmuramoyl-l-alanine amidase (AM) and endo-β-N-acetylglucosaminidase (GL) have been in the research focus for long time. This review addresses phenotypic consequences of atl mutants, impact of Atl in virulence, the mechanism of targeting to the septum region, regulation of atl, the structure of the amidase and the repeat regions, as well as the phylogeny of Atl and its use in Staphylococcus genus and species typing.

Genome Instability at Common Fragile Sites: Searching for the Cause of Their Instability

Common fragile sites (CFS) are heritable nonrandomly distributed loci on human chromosomes that exhibit an increased frequency of chromosomal breakage under conditions of replication stress. They are considered the preferential targets for high genomic instability from the earliest stages of human cancer development, and increased chromosome instability at these loci has been observed following replication stress in a subset of human genetic diseases. Despite their biological and medical relevance, the molecular basis of CFS fragility in vivo has not been fully elucidated. At present, different models have been proposed to explain how instability at CFS arises and multiple factors seem to contribute to their instability. However, all these models involve DNA replication and suggest that replication fork stalling along CFS during DNA synthesis is a very frequent event. Consistent with this, the maintenance of CFS stability relies on the ATR-dependent checkpoint, together with a number of proteins promoting the recovery of stalled replication forks. In this review, we discuss mainly the possible causes that threaten the integrity of CFS in the light of new findings, paying particular attention to the role of the S-phase checkpoint.

Specific DNA-RNA Hybrid Recognition by TAL Effectors

The transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of theDNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections.

A quantitative model of the initiation of DNA replication in Saccharomyces cerevisiae predicts the effects of system perturbations

BackgroundEukaryotic cell proliferation involves DNA replication, a tightly regulated process mediated by a multitude of protein factors. In budding yeast, the initiation of replication is facilitated by the heterohexameric origin recognition complex (ORC). ORC binds to specific origins of replication and then serves as a scaffold for the recruitment of other factors such as Cdt1, Cdc6, the Mcm2-7 complex, Cdc45 and the Dbf4-Cdc7 kinase complex. While many of the mechanisms controlling these associations are well documented, mathematical models are needed to explore the network’s dynamic behaviour. We have developed an ordinary differential equation-based model of the protein-protein interaction network describing replication initiation.ResultsThe model was validated against quantified levels of protein factors over a range of cell cycle timepoints. Using chromatin extracts from synchronized Saccharomyces cerevisiae cell cultures, we were able to monitor the in vivo fluctuations of several of the aforementioned proteins, with additional data obtained from the literature. The model behaviour conforms to perturbation trials previously reported in the literature, and accurately predicts the results of our own knockdown experiments. Furthermore, we successfully incorporated our replication initiation model into an established model of the entire yeast cell cycle, thus providing a comprehensive description of these processes.ConclusionsThis study establishes a robust model of the processes driving DNA replication initiation. The model was validated against observed cell concentrations of the driving factors, and characterizes the interactions between factors implicated in eukaryotic DNA replication. Finally, this model can serve as a guide in efforts to generate a comprehensive model of the mammalian cell cycle in order to explore cancer-related phenotypes.

Unexpected Role for Helicobacter pylori DNA Polymerase I As a Source of Genetic Variability

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity.

Prognosis Is Genetically Fixed In Polycythemia Vera and Definable by Gene Expression Profiling

AbstractAbstract 1987Polycythemia vera (PV) is a chronic, clonal, stem cell disorder whose natural history is usually indolent but an undefined fraction of patients have a clinical course characterized by extramedullary hematopoiesis, myelofibrosis, massive splenomegaly, bone marrow failure, and a propensity to develop spontaneous acute leukemia or early death. Recently, a prognostic scoring system was developed to determine the survival of such patients after the onset of bone marrow failure (Blood, 2008; 111:3383-7). However, at that point, for most such patients, only palliative therapy is available. We have been conducting a gene expression profiling (GEP) study in PV peripheral blood (pb) CD34+ cells using oligonucleotide microarray technology, confirmed by QPCR, and now report that in patients who develop an aggressive form of PV, the associated genetic abnormalities are time independent, providing an opportunity to identify patients at risk before bone marrow failure ensues. Our observations were obtained in a cross-sectional study of 19 PV patients (8 men and 11 women) from whom pb CD34+ cells were harvested for GEP in comparison to immunophenotypically similar G-CSF mobilized pb CD34+cells from normal individuals (3 men and 3 women). The median age (55 years; range 33–71), disease duration (11.5 years; range 1–33) and JAK2 V617F allele burden (92%: range 50–100) of the men at the time of study were not significantly different from the women (median age 51 years; range 42–70); disease duration, (8 years; range 1–13) and JAK2 V617F allele burden (100%; range 49–100);16 patients were homozygous for the mutation. Gender-specific differences in PV clinical features (Haematologica 2010; 95:1090-7) were mirrored by gender-specific differences in GEP between the men and women patients, allowing us to identify a core set of 89 genes (31 down regulated and 58 up regulated) shared in common. Using this core gene set and unsupervised hierarchical clustering, the 19 PV patients could be segregated into a small homogeneous group (6 patients; 3 men and 3 women) and a larger heterogeneous group(5 men and 8 women), which did not differ with respect to median age (64.5 vs 67 years); median disease duration (10.5; range 5–20, vs 7 years; range 1–33); JAK2 V617F allele burden (100% vs 88%), or leukocyte and platelet counts, but did differ with respect to their clinical characteristics with the smaller group having a lower median hemoglobin level (10.7 gm% vs 13.1 gm%; p= 0.01), a greater number of thrombotic events (p = 0.017), greater spleen size, (p= 0.01), a need for splenectomy (p= 0.017), a need for chemotherapy (p =0.009), and a shorter median survival time (17 years vs 25 years; log rank p value 0.02). Since the median disease duration of the two groups did not differ, the underlying genetic abnormalities must be time independent. Analysis of the core gene set revealed a “stromal gene signature” consisting of 16 matricellular and collagen genes, which, when subjected to unsupervised hierarchical clustering, not only led to the same group distinctions but resolved the genetic heterogeneity of the larger group. Remarkably, the stromal genes were down regulated in the more aggressive group and up regulated in the more indolent one. To determine the overall genetic differences between these two clinically distinct groups, we used Gene Set Enrichment Analysis to identify the top 50 up or down regulated genes in each. Among the notable genes down regulated in the indolent group were BAALC, DACH1, CD44, DDX17 and VIM, and in the aggressive group, FOXO1, IRF8, ABCD1, HOXA5, SOX4 and CRIM1. In the indolent group, LSM1, FADS2, CDKN1A, E2F1, MAZ and FHL2 were up regulated. FHL2 was also the only top 50 up regulated gene shared in common with the aggressive group, and one of the top two most up regulated genes in the patient population as a whole, LSM1 being the other, emphasizing the importance of these genes in disease pathogenesis. Equally important, in the aggressive group, there was an up regulated 17 gene signature involving DNA replication unique to it alone (p = 0.032). These data solidify the contention that despite the commonality of JAK2 V617F, PV is not a monolithic illness, that prognosis is independent of disease duration, provide insight into the pathogenesis of PV and offer the possibility of genetically identifying those patients most at risk of disease-associated morbidity and mortality when they can still benefit from therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Activation of guanine-β- d-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

The deoxyguanosine (GdR) analog guanine-ß- d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

Acetylation of WRN Protein Regulates Its Stability by Inhibiting Ubiquitination

BackgroundWRN is a multi-functional protein involving DNA replication, recombination and repair. WRN acetylation has been demonstrated playing an important role in response to DNA damage. We previously found that WRN acetylation can regulate its enzymatic activities and nuclear distribution.Methodology/Principal FindingHere, we investigated the factors involved in WRN acetylation and found that CBP and p300 are the only major acetyltransferases for WRN acetylation. We further identified 6 lysine residues in WRN that are subject to acetylation. Interestingly, WRN acetylation can increase its protein stability. SIRT1-mediated deacetylation of WRN reverses this effect. CBP dramatically increases the half-life of wild type WRN, while mutation of these 6 lysine residues (WRN-6KR) abrogates this increase. We further found that WRN stability is regulated by the ubiquitination pathway and WRN acetylation by CBP significantly reduces its ubiquitination. Importantly, we found that WRN is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells stably expressing WRN-6KR, which mimics unacetylated WRN, display significantly higher MMC sensitivity compared with the cells expressing wild-type WRN.Conclusion/SignificanceTaken together, these data demonstrate that WRN acetylation regulates its stability and has significant implications regarding the role of acetylation on WRN function in response to DNA damage.

Multiple Uses of Lys63-Polyubiquitination in the Ciliate Sterkiella histriomuscorum

Poly-ubiquitination through Lys63, a post-translational modification system, is emerging as a mechanism implicated in a variety of non-proteolytic functions in eukaryotic cells. This modification is mainly carried out via a unique complex formed between a canonical ubiquitin-conjugating enzyme (E2), Ubc13, and an E2-variant, Uev. To date, a wealth of functional information has been obtained mostly from animals and yeast. Here, we show that the ciliate Sterkiella histriomuscorum harbors a Ubc13 gene that encodes a putative protein exhibiting 60-64% identity with Ubc13 from organisms of diverse eukaryotic phyla. A yeast double-hybrid assay and 3D-structure modelling gave evidence that ShUbc13 is able to physically interact with ShUev; the existence of such a complex in a ciliate attests for its ancient origin. Western blotting reveals that Ubc13 is present in different stages of the vegetative cycle of S. histriomuscorum. Immunolocalization of Ubc13 and K63 polyubiquitinated proteins shows three main locations, namely the nucleus, cilia and plasma membrane. Possible functions of ShUbc13 and K63 polyubiquitination in this single-celled organism are considered with reference to current knowledge. In particular their likely involvement in DNA replication and DNA damage response is especially discussed.

Chemo-Mechanical Study of a Hexameric Helicase on the Single-Molecule Level

Cell division involves DNA replication that requires the opening of double stranded DNA by cellular machines known as helicases. Although many of those essential enzymes are of hexameric structure, not much is known about coordination and the mechano-chemical function of these multimeric enzymes. Here, we study with single-molecule FRET (Forster resonance energy transfer) a hexameric helicase of the DnaB family, called G40P. DnaB helicase is the essential replication helicase in prokaryotes and consists of 6 identical subunits that exhibit the widely shared RecA-fold in biological enzymes. In order to gain information about the chemo-mechanical cycle of G40P, we followed the time trajectory of individual enzymes while unwinding a DNA duplex. By the addition of the non-hydrolyzable ATP analogue ATPgS to the reaction at low ratio of ATPgS to ATP, we observed significant stalls during the unwinding process. Varying the concentration of ATPgS did not affect the lifetime of the stall, which indicates a strong coordination between the identical subunits. Based on this observation, we propose a highly coordinated subsequent ATP hydrolysis between the subunits, where binding ATPgS at a single site can stall the entire helicase. Furthermore, under suboptimal conditions like low ATP concentrations, we observed frequent repetitive slippage events of individual helicases, indicating a transient loss of tight binding to the DNA substrate.

Involvement of a chromatin remodeling complex in damage tolerance during DNA replication

ATP-dependent chromatin remodeling complexes have been shown to participate in DNA replication in addition to transcription and DNA repair. However, the mechanisms of their involvement in DNA replication remain unclear. Here, we reveal a specific function of the yeast INO80 chromatin remodeling complex in the DNA damage tolerance pathways. Whereas INO80 is necessary for the resumption of replication at forks stalled by methyl methane sulfonate (MMS), it is not required for replication fork collapse after treatment with hydroxyurea (HU). Mechanistically, INO80 regulates DNA damage tolerance during replication through modulation of PCNA (proliferating cell nuclear antigen) ubiquitination and Rad51-mediated processing of recombination intermediates at impeded replication forks. Our findings establish a mechanistic link between INO80 and DNA damage tolerance pathways, indicating that chromatin remodeling is important for accurate DNA replication.

Mechanism of Werner DNA Helicase: POT1 and RPA Stimulates WRN to Unwind beyond Gaps in the Translocating Strand

WRN belongs to the RecQ family of DNA helicases and it plays a role in recombination, replication, telomere maintenance and long-patch base excision repair. Here, we demonstrate that WRN efficiently unwinds DNA substrates containing a 1-nucleotide gap in the translocating DNA strand, but when the gap size is increased to 3-nucleotides unwinding activity significantly declines. In contrast, E. coli UvrD (3′→5′ helicase), which recognizes nicks in DNA to initiate unwinding, does not unwind past a 1-nucleotide gap. This unique ability of WRN to bypass gaps supports its involvement in DNA replication and LP-BER where such gaps can be produced by glycosylases and the apurinic/apyrimidinic endonuclease 1 (APE1). Furthermore, we tested telomere repeat binding factor 2 (TRF2), both variants 1 and 2 of protector of telomeres 1 (POT1v1 and POT1v2) and RPA on telomeric DNA substrates containing much bigger gaps than 3-nucleotides in order to determine whether unwinding could be facilitated through WRN-protein interaction. Interestingly, POT1v1 and RPA are capable of stimulating WRN helicase on gapped DNA and 5′-overhang substrates, respectively.

Involvement of DNA replication in phage lambda Red‐mediated homologous recombination

Crosses between a non-replicating linear bacteriophage lambda chromosome and a replicating plasmid bearing a short cloned segment of lambda DNA were monitored by extracting DNA from infected cells, and analysing it via restriction endonuclease digestion and Southern blots. Recombinant formation resulting from the action of the Red homologous recombination system, observed directly in this way, was found to be fast, efficient, independent of the bacterial recA function and highly dependent upon replication of the target plasmid. These features of the experimental system faithfully model Red-mediated recombination in a lytically infected cell in which phage DNA replication is occurring. Neither of the previously established mechanisms by which the Red system can operate--strand annealing or strand invasion--accounts well for these findings. A third mechanism, replisome invasion, involving replication directly in the recombination mechanism, is invoked as an alternative.

Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair.

Expansions of CAG repeat tracts in the germ line underlie several neurological diseases. In human patients and mouse models, CAG repeat tracts display an ongoing instability in neurons, which may exacerbate disease symptoms. It is unclear how repeats are destabilized in nondividing cells, but it cannot involve DNA replication. We showed previously that transcription through CAG repeats induces their instability (Y. Lin, V. Dion, and J. H. Wilson, Nat. Struct. Mol. Biol. 13:179-180). Here, we present a genetic analysis of the link between transcription-induced repeat instability and nucleotide excision repair (NER) in human cells. We show that short interfering RNA-mediated knockdown of CSB, a component specifically required for transcription-coupled NER (TC-NER), and knockdowns of ERCC1 and XPG, which incise DNA adjacent to damage, stabilize CAG repeat tracts. These results suggest that TC-NER is involved in the pathway for transcription-induced CAG repeat instability. In contrast, knockdowns of OGG1 and APEX1, key components involved in base excision repair, did not affect repeat instability. In addition, repeats are stabilized by knockdown of transcription factor IIS, consistent with a requirement for RNA polymerase II (RNAPII) to backtrack from a transcription block. Repeats also are stabilized by knockdown of either BRCA1 or BARD1, which together function as an E3 ligase that can ubiquitinate arrested RNAPII. Treatment with the proteasome inhibitor MG132, which stabilizes repeats, confirms proteasome involvement. We integrate these observations into a tentative pathway for transcription-induced CAG repeat instability that can account for the contractions observed here and potentially for the contractions and expansions seen with human diseases.

GTPase Activity of Mycobacterial FtsZ Is Impaired Due to Its Transphosphorylation by the Eukaryotic-type Ser/Thr Kinase, PknA

FtsZ, a homolog of eukaryotic tubulin, is involved in the process of cell division, particularly in septum formation in bacteria. The primary amino acid sequences of this protein are fairly conserved in prokaryotes. We observed that a eukaryotic-type Ser/Thr protein kinase, PknA from Mycobacterium tuberculosis, when expressed in Escherichia coli exhibited cell elongation due to a defect in septum formation. We found that FtsZ either from Escherichia coli (eFtsZ) or from M. tuberculosis (mFtsZ) was phosphorylated on co-expression with PknA. Consistent with these observations, solid phase binding and in vitro kinase assays revealed the ability of PknA to interact with mFtsZ protein and also to phosphorylate it. We, therefore, ascertained mFtsZ as a substrate of PknA. Furthermore, the phosphorylated mFtsZ exhibited impairment in its GTP hydrolysis and polymerization abilities. Thus, our results highlighted the ability of PknA to phosphorylate as well as to regulate the functionality of FtsZ, the protein central to cell division throughout the bacterial lineage.

Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints

Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression.

A new molecular model of cellular aging based on Werner syndrome

In the Hayflick model, a decrease in the number of cells capable of undergoing proliferation constitutes the main criterion of cellular aging and is closely linked to organismal aging. Evidence suggests that a reduction of DNA replication capacity or a failure in the regulation systems of DNA replication occurs in aging cells, which leads to cellular replicative senescence. DNA replication depends on two parameters: the number of active replicons and the rate of chain elongation. Epigenetic parameters, in particular methylation, would be able to, either directly or indirectly, regulate replication origin activity of normal mammalian cells, as well as subsequent DNA replication. Werner syndrome (WS) is an autosomal recessive disorder that results in premature aging and is considered to be a model system for the study of cellular senescence and aging. WRN could involve DNA replication initiation, replication foci establishment, and the resolution of stalled replication forks during replication. In this paper, a molecular model of in vitro cellular aging is presented in which changes in DNA methylation, in particular, global hypomethylation related to methyltransferase Dnmt1 downregulation, and specific hypermethylation related to methyltransferase Dnmt3b upregulation as seen during cellular aging, could be responsible for the inactivation of replication origins or foci and the subsequent documented reduction in DNA replication capacity and increased mutations that are observed in senescent cells. Thus, Werner syndrome cells could be mimicking what is observed in normal aging in an accelerated form.

Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication

Chromatin is suppressive in nature to cellular enzymes that metabolize DNA, mainly due to the inherent inaccessibility of the DNA template. Despite extensive understanding of the involvement of chromatin-modifying factors in transcription, roles of related activities in DNA replication remain largely elusive. Here, we show that the heterodimeric transcriptional elongation factor FACT (facilitates chromatin transcription) is functionally linked to DNA synthesis. Its involvement in DNA replication is partly mediated by the stable association with the replicative helicase complex, MCM, and further by the coexistence with MCM on replication origin. Furthermore, relying on its nucleosome-reorganizing activity, FACT can facilitate chromatin unwinding by the MCM complex, which is otherwise inert on the nucleosomal template. As a consequence, the physical and functional interaction between FACT and MCM is an important determinant in the proper initiation of DNA replication and S phase in vivo. Together, our findings identify FACT as an integral and conserved component of the endogenous replication machinery, and support a model in which the concerted action of helicase and chromatin-modifying activities promotes chromosome replication.

Involvement of DNA replication in ultraviolet-induced apoptosis of mammalian cells

Exposure of cells to ultraviolet (UV) light damages the genome and the persistence of DNA lesions triggers apoptosis in mammalian cells. RNA transcription blockage by DNA damage is believed to be implicated in signaling for UV-induced apoptosis, but the role played by DNA replication in this process is still unclear. To address this point, we have employed the DNA polymerase inhibitor aphidicolin in UV-irradiated wild-type and XPB-mutated Chinese hamster ovary cells. The data obtained with synchronized cells indicate that induction of apoptosis by UV light is independent of the cell cycle phase. Nevertheless, cells treated with aphidicolin after UV exposure showed a significant prevention of apoptosis induction when compared to proliferating cells. These results were observed in both DNA-repair proficient and deficient cells, indicating that the prevention of apoptosis by aphidicolin is independent of the cells' ability to repair the photolesions caused by UV. Taken together, these data suggest that replication of damaged DNA also leads to critical events signaling for UV-induced cell death.