RESUMO
DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.
Assuntos
Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Bactérias/metabolismo , Instabilidade Cromossômica/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Instabilidade Genômica , Humanos , Proteínas de Membrana Transportadoras/fisiologia , Mutagênese , Mutação , Fatores de Transcrição/metabolismoRESUMO
Microbes and human cells possess mechanisms of mutagenesis activated by stress responses. Stress-inducible mutagenesis mechanisms may provide important models for mutagenesis that drives host-pathogen interactions, antibiotic resistance, and possibly much of evolution generally. In Escherichia coli, repair of DNA double-strand breaks is switched to a mutagenic mode, using error-prone DNA polymerases, via the SOS DNA damage and general (σS) stress responses. We investigated small RNA (sRNA) clients of Hfq, an RNA chaperone that promotes mutagenic break repair (MBR), and found that GcvB promotes MBR by allowing a robust σS response, achieved via opposing the membrane stress (σE) response. Cells that lack gcvB were MBR deficient and displayed reduced σS-dependent transcription but not reduced σS protein levels. The defects in MBR and σS-dependent transcription in ΔgcvB cells were alleviated by artificially increasing σS levels, implying that GcvB promotes mutagenesis by allowing a normal σS response. ΔgcvB cells were highly induced for the σE response, and blocking σE response induction restored both mutagenesis and σS-promoted transcription. We suggest that GcvB may promote the σS response and mutagenesis indirectly, by promoting membrane integrity, which keeps σE levels lower. At high levels, σE might outcompete σS for binding RNA polymerase and so reduce the σS response and mutagenesis. The data show the delicate balance of stress response modulation of mutagenesis. IMPORTANCE: Mutagenesis mechanisms upregulated by stress responses promote de novo antibiotic resistance and cross-resistance in bacteria, antifungal drug resistance in yeasts, and genome instability in cancer cells under hypoxic stress. This paper describes the role of a small RNA (sRNA) in promoting a stress-inducible-mutagenesis mechanism, mutagenic DNA break repair in Escherichia coli The roles of many sRNAs in E. coli remain unknown. This study shows that ΔgcvB cells, which lack the GcvB sRNA, display a hyperactivated membrane stress response and reduced general stress response, possibly because of sigma factor competition for RNA polymerase. This results in a mutagenic break repair defect. The data illuminate a function of GcvB sRNA in opposing the membrane stress response, and thus indirectly upregulating mutagenesis.
Assuntos
Aminoácido Oxirredutases/metabolismo , Membrana Celular/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagênicos/toxicidade , Pequeno RNA não Traduzido/metabolismo , Aminoácido Oxirredutases/genética , Membrana Celular/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Pequeno RNA não Traduzido/genética , Fator sigma/genética , Fator sigma/metabolismoRESUMO
There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ'χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-C(tag)) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except ß) from this strain contained one molecule of γ-C(tag) per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ'χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F' episome.
Assuntos
DNA Polimerase III/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Holoenzimas/metabolismo , DNA Polimerase III/química , DNA Polimerase III/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Citometria de Fluxo , Dosagem de Genes , Holoenzimas/química , Holoenzimas/genética , Immunoblotting , Viabilidade Microbiana/genética , Mutação , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
With the wide availability of whole-genome sequencing (WGS), genetic mapping has become the rate-limiting step, inhibiting unbiased forward genetics in even the most tractable model organisms. We introduce a rapid deconvolution resource and method for untagged causative mutations after mutagenesis, screens, and WGS in Escherichia coli. We created Deconvoluter-ordered libraries with selectable insertions every 50 kb in the E. coli genome. The Deconvoluter method uses these for replacement of untagged mutations in the genome using a phage-P1-based gene-replacement strategy. We validate the Deconvoluter resource by deconvolution of 17 of 17 phenotype-altering mutations from a screen of N-ethyl-N-nitrosourea-induced mutants. The Deconvoluter resource permits rapid unbiased screens and gene/function identification and will enable exploration of functions of essential genes and undiscovered genes/sites/alleles not represented in existing deletion collections. This resource for unbiased forward-genetic screens with mapping-by-sequencing ('forward genomics') demonstrates a strategy that could similarly enable rapid screens in many other microbes.
Assuntos
Escherichia coli/genética , Biblioteca Gênica , Genoma Bacteriano , Genômica/métodos , Mutagênese Insercional/métodos , Mutação , Algoritmos , Bacteriófago P1/genética , Escherichia coli/efeitos dos fármacos , Etilnitrosoureia/farmacologia , Genótipo , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.
Assuntos
Cromossomos Bacterianos/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Virais/genética , Animais , Bacteriófago mu/química , Cromossomos Bacterianos/química , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/química , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Autoantígeno Ku , Camundongos , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biologia Sintética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Proteínas Virais/metabolismoRESUMO
Mechanisms of DNA repair and mutagenesis are defined on the basis of relatively few proteins acting on DNA, yet the identities and functions of all proteins required are unknown. Here, we identify the network that underlies mutagenic repair of DNA breaks in stressed Escherichia coli and define functions for much of it. Using a comprehensive screen, we identified a network of ≥93 genes that function in mutation. Most operate upstream of activation of three required stress responses (RpoS, RpoE, and SOS, key network hubs), apparently sensing stress. The results reveal how a network integrates mutagenic repair into the biology of the cell, show specific pathways of environmental sensing, demonstrate the centrality of stress responses, and imply that these responses are attractive as potential drug targets for blocking the evolution of pathogens.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Estresse Fisiológico/genética , Proteínas de Bactérias/genética , Mutagênese/genética , Resposta SOS em Genética/genética , Fator sigma/genéticaRESUMO
Evolutionary theory assumed that mutations occur constantly, gradually, and randomly over time. This formulation from the "modern synthesis" of the 1930s was embraced decades before molecular understanding of genes or mutations. Since then, our labs and others have elucidated mutation mechanisms activated by stress responses. Stress-induced mutation mechanisms produce mutations, potentially accelerating evolution, specifically when cells are maladapted to their environment, that is, when they are stressed. The mechanisms of stress-induced mutation that are being revealed experimentally in laboratory settings provide compelling models for mutagenesis that propels pathogen-host adaptation, antibiotic resistance, cancer progression and resistance, and perhaps much of evolution generally. We discuss double-strand-break-dependent stress-induced mutation in Escherichia coli. Recent results illustrate how a stress response activates mutagenesis and demonstrate this mechanism's generality and importance to spontaneous mutation. New data also suggest a possible harmony between previous, apparently opposed, models for the molecular mechanism. They additionally strengthen the case for anti-evolvability therapeutics for infectious disease and cancer.
Assuntos
Anti-Infecciosos/farmacologia , Escherichia coli/genética , Evolução Molecular , Mutagênese , Estresse Fisiológico , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Bacteriano/genética , Desenho de Fármacos , Resistência Microbiana a Medicamentos/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/fisiologia , Humanos , Seleção GenéticaAssuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Adaptação Fisiológica , Antibacterianos/análise , Técnicas Bacteriológicas , Ciprofloxacina/análise , Meios de Cultura , DNA Girase/genética , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Evolução Molecular , Técnicas Analíticas Microfluídicas , Movimento , Mutagênese , Fenótipo , Estresse FisiológicoRESUMO
Previous work showed that about 85% of stress-induced mutations associated with DNA double-strand break repair in carbon-starved Escherichia coli result from error-prone DNA polymerase IV (Pol IV) (DinB) and that the mutagenesis is controlled by the RpoS stress response, which upregulates dinB. We report that the remaining mutagenesis requires high-fidelity Pol II, and that this component also requires RpoS. The results identify a second DNA polymerase contributing to stress-induced mutagenesis and show that RpoS promotes mutagenesis by more than the simple upregulation of dinB.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Fator sigma/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Bactérias/genética , DNA Polimerase II/genética , DNA Polimerase beta/genética , Reparo do DNA/genética , Proteínas de Escherichia coli/genética , Mutagênese , Mutação/genética , Fator sigma/genética , Transdução de Sinais/genéticaRESUMO
The nitrogen assimilation control protein (NAC) of Klebsiella pneumoniae is a LysR-type transcriptional regulator that activates transcription when bound to a DNA site (ATAA-N5-TnGTAT) centered at a variety of distances from the start of transcription. The NAC-binding site from the hutU promoter (NBShutU) is centered at -64 relative to the start of transcription but can activate the lacZ promoter from sites at -64, -54, -52, and -42 but not from sites at -47 or -59. However, the NBSs from the ureD promoter (ureDp) and codB promoter (codBp) are centered at -47 and -59, respectively, and NAC is fully functional at these promoters. Therefore, we compared the activities of the NBShutU and NBSureD within the context of ureDp as well as within codBp. The NBShutU functioned at both of these sites. The NBSureD has the same asymmetric core as the NBShutU. Inverting the NBSureD abolished more than 99% of NAC's ability to activate ureDp. The key to the activation lies in the TnG segment of the TnGTAT half of the NBSureD. Changing TnG to GnT, TnT, or GnG drastically reduced ureDp activation (to 0.5%, 6%, or 15% of wild-type activation, respectively). The function of the NBSureD, like that of the NBShutU, requires that the TnGTAT half of the NBS be on the promoter-proximal (downstream) side of the NBS. Taken together, our data suggest that the positional specificity of an NBS is dependent on the promoter in question and is more flexible than previously thought, allowing considerable latitude both in distance and on the face of the DNA helix for the NBS relative to that of RNA polymerase.
Assuntos
Proteínas de Bactérias/metabolismo , Klebsiella pneumoniae/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Klebsiella pneumoniae/genética , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/citologia , Escherichia coli/enzimologia , Estresse Fisiológico , Quebras de DNA de Cadeia Dupla , DNA Polimerase I/química , DNA Polimerase I/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação da Fase de Leitura/genética , Modelos Biológicos , Mutagênese/genética , Estrutura Terciária de Proteína , Resposta SOS em Genética/genética , Serina Endopeptidases/metabolismoRESUMO
Most LysR-type transcriptional regulators (LTTRs) function as tetramers when regulating gene expression. The nitrogen assimilation control protein (NAC) generally functions as a dimer when binding to DNA and activating transcription. However, at some sites, NAC binds as a tetramer. Like many LTTRs, NAC tetramers can recognize sites with long footprints (74 bp for the site at nac) with a substantial DNA bend or short footprints (56 bp for the site at cod) with less DNA bending. However, unlike other LTTRs, NAC can recognize both types of sites in the absence of physiologically relevant coeffectors, suggesting that the two conformers of the NAC tetramer (extended and compact) are interchangeable without the need for any modification to induce or stabilize the change. In order for NAC to bind as a tetramer, three interactions must exist: an interaction between the two NAC dimers and an interaction between each NAC dimer and its corresponding binding site. The interaction between one dimer and its DNA site can be weak (recognizing a half-site rather than a full dimer-binding site), but the other two interactions must be strong. Since the conformation of the NAC tetramer (extended or compact) is determined by the nature of the DNA site without the intervention of a small molecule, we argue that the coeffector that determines the conformation of the NAC tetramer is the DNA site to which it binds.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fatores de Transcrição/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genéticaRESUMO
Klebsiella pneumoniae is able to utilize many nitrogen sources, and the utilization of some of these nitrogen sources is dependent on the nitrogen assimilation control (NAC) protein. Seven NAC-regulated promoters have been characterized in K. pneumoniae, and nine NAC-regulated promoters have been found by microarray analysis in Escherichia coli. So far, all characterized NAC-regulated promoters have been directly related to nitrogen metabolism. We have used a genome-wide analysis of NAC binding under nitrogen limitation to identify the regions of the chromosome associated with NAC in K. pneumoniae. We found NAC associated with 99 unique regions of the chromosome under nitrogen limitation. In vitro, 84 of the 99 regions associate strongly enough with purified NAC to produce a shifted band by electrophoretic mobility shift assay. Primer extension analysis of the mRNA from genes associated with 17 of the fragments demonstrated that at least one gene associated with each fragment was NAC regulated under nitrogen limitation. The large size of the NAC regulon in K. pneumoniae indicates that NAC plays a larger role in the nitrogen stress response than it does in E. coli. Although a majority of the genes with identifiable functions that associated with NAC under nitrogen limitation are involved in nitrogen metabolism, smaller subsets are associated with carbon and energy acquisition (18 genes), and growth rate control (10 genes). This suggests an expanded role for NAC regulation during the nitrogen stress response, where NAC not only regulates genes involved in nitrogen metabolism but also regulates genes involved in balancing carbon and nitrogen pools and growth rate.
Assuntos
Proteínas de Bactérias/metabolismo , Klebsiella pneumoniae/metabolismo , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Imunoprecipitação da Cromatina , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica , Imunoprecipitação , Klebsiella pneumoniae/genética , Proteínas PII Reguladoras de Nitrogênio/genética , Reação em Cadeia da Polimerase , Regulon/genética , Regulon/fisiologia , Fatores de Transcrição/genéticaRESUMO
Plasmid-based collections of fluorescent protein fusions are valuable and versatile resources, facilitating systematic studies of protein localization in multiple genetic backgrounds. At present, however, few such collections exist for the analysis of protein localization in any organism. To address this deficiency, we present here a plasmid-based set of resources for the analysis of protein localization in the budding yeast. Specifically, we constructed a suite of low-copy destination vectors for recombination-based cloning of yeast genes as fluorescent protein fusions. We cloned a set of 384 yeast genes encoding kinases, transcription factors and signaling proteins as "recombination-ready" cassettes; by Gateway cloning, these genes with native promoters can be easily introduced into the destination vectors described above, generating carboxy-terminal fusions to fluorescent proteins. Using these reagents, we constructed a subcollection of 276 genes encoding carboxy-terminal fusions to yellow fluorescent protein (vYFP). This collection encompasses 14 autophagy-related (ATG) genes, and we localized these Atgp-vYFP chimeras during rapamycin-induced autophagy. To illustrate further the utility of this collection as a tool in exploring the functions and interactions of proteins in a pathway, we localized a subset of these Atg-vYFP chimeras in a strain deleted for the scaffolding protein Atg11p. In addition, we validated previous results identifying the integral membrane protein Atg9p at the pre-autophagosomal structure upon overexpression of ATG11 and upon deletion of ATG1. Collectively, this plasmid-based resource of yeast gene-vYFP fusions provides an initial toolkit for a variety of systematic and large-scale localization studies exploring pathway biology in the budding yeast.
Assuntos
Autofagia/fisiologia , Proteínas Luminescentes/metabolismo , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Antifúngicos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas Luminescentes/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/metabolismoRESUMO
During G1 phase, a prereplicative complex (pre-RC) that determines where DNA synthesis initiates forms at origins. The Sir2p histone deacetylase inhibits pre-RC assembly at a subset of origins, suggesting that Sir2p inhibits DNA replication through a unique aspect of origin structure. Here, we identified five SIR2-sensitive origins on chromosomes III and VI. Linker scan analysis of two origins indicated that they share a common organization, including an inhibitory sequence positioned 3' to the sites of origin recognition complex (ORC) binding and pre-RC assembly. This inhibitory sequence (I(S)) required SIR2 for its activity, suggesting that SIR2 inhibits origins through this sequence. Furthermore, I(S) elements occurred within positioned nucleosomes, and Abf1p-mediated exclusion of nucleosomes from the origin abrogated the inhibition. These data suggest that Sir2p and I(S) elements inhibit origin activity by promoting an unfavorable chromatin structure for pre-RC assembly.