RESUMO
We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin-hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin-hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.
Assuntos
Núcleo Celular/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Compartimento Celular , Nucléolo Celular , Núcleo Celular/química , DNA de Cadeia Simples , Proteínas de Ligação a DNA/isolamento & purificação , Resposta ao Choque Térmico , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Matriz Nuclear , Conformação de Ácido Nucleico , Fosfoproteínas/isolamento & purificação , Ligação Proteica , Proteínas de Ligação a RNA/isolamento & purificação , Origem de Replicação , Proteína de Replicação A , Células Tumorais Cultivadas , NucleolinaRESUMO
The mechanism by which DNA helicases unwind DNA was tested; an "unwinding complex" between the SV40 large tumor antigen (T antigen) and a DNA molecule designed to resemble a replication fork was probed. In an adenosine triphosphate (ATP)-dependent reaction, T antigen quantitatively recognized this synthetic replication fork and bound the DNA primarily as a hexamer. The T antigen bound only one of the two strands at the fork, an asymmetric interaction consistent with the 3'----5' directionality of the DNA helicase activity of T antigen. Binding to chemically modified DNA substrates indicated that the DNA helicase recognized the DNA primarily through the sugar-phosphate backbone. Ethylation of six top strand phosphates at the junction of single-stranded and double-stranded DNA inhibited the DNA helicase activity of T antigen. Neither a 3' single-stranded end on the DNA substrate nor ATP hydrolysis was required for T antigen to bind the replication fork. These data suggest that T antigen can directly bind the replication fork through recognition of a fork-specific structure.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Replicação do DNA/imunologia , Trifosfato de Adenosina/farmacologia , DNA Helicases/fisiologia , DNA de Cadeia Simples/metabolismo , Dietil Pirocarbonato/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Etilnitrosoureia/farmacologia , Formiatos/farmacologia , Permanganato de Potássio/farmacologia , Ésteres do Ácido Sulfúrico/farmacologia , Fatores de TempoRESUMO
Nucleolin is a c-Myc-induced gene product with defined roles in ribosomal RNA processing and the inhibition of chromosomal DNA replication following stress. Here we find that changes in nucleolin protein levels in unstressed cells cause parallel changes in the amount of p53 protein. Alterations in p53 levels arise from nucleolin binding to the p53 antagonist Hdm2, resulting in the inhibition of both p53 ubiquitination and Hdm2 auto-ubiquitination. Nucleolin does not alter p53 ubiquitination by human papillomavirus E6, indicating that the effect is specific for Hdm2. Although the inhibition of ligase activity would be expected to stabilize Hdm2, we instead find that nucleolin also reduces Hdm2 protein levels, demonstrating that nucleolin inhibits Hdm2 using multiple mechanisms. Increases in nucleolin levels in unstressed cells led to higher expression of p21(cip1/waf1), a reduced rate of cellular proliferation, and an increase in apoptosis. Thus, nucleolin has a number of properties in common with the tumor suppressor ARF (alternate reading frame). We propose that nucleolin, like ARF, responds to hyperproliferative signals by upregulation of p53 through Hdm2 inhibition.
Assuntos
Fosfoproteínas/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas de Ligação a RNA/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Western Blotting , Linhagem Celular , Regulação para Baixo , Meia-Vida , Humanos , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Ubiquitina/metabolismo , NucleolinaRESUMO
Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.
Assuntos
Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Bases , Sítios de Ligação , Divisão Celular , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Humanos , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/metabolismo , Proteína de Replicação A , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.
Assuntos
Replicação do DNA , DNA Viral/ultraestrutura , Regulação Viral da Expressão Gênica , Proteínas/fisiologia , Origem de Replicação , Vírus 40 dos Símios/genética , Transativadores , Sequência de Bases , DNA Helicases/metabolismo , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Desnaturação de Ácido NucleicoRESUMO
Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Bases , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão e Varredura , Dados de Sequência Molecular , Proteínas Nucleares , Fosforilação , Ligação Proteica , Conformação Proteica , Proteína de Replicação ARESUMO
The rate of open promoter complex formation was measured on lac ps promoter DNA templates differing in negative superhelicity. The templates ranged from fully relaxed to those with numbers of superhelical turns exceeding that of form I plasmid DNA. The observed transcription response had two clearly distinguished phases: an initial rapid rise in rate followed eventually by a precipitous inhibition. The stimulation phase involved a nearly 40-fold increase in rate, which peaks at superhelical densities near that of isolated form I plasmid DNA. The introduction of more negative superhelical turns leads to inhibition. The magnitude of the response and the observation of both increases and decreases suggest that minor differences in superhelicity in vivo could lead to significant increases or decreases in transcription rate. The increase in rate was found to be directly proportional to the free energy of supercoiling; that is, to the square of the superhelical density. We suggest that the energy may be used both for enhanced DNA melting and for changes in DNA structure that alter the helical "face" with which RNA polymerase must interact. A quantitative method is presented that allows simple estimation of differences in the supercoiling response among promoters, both in the presence and in the absence of added factors.
Assuntos
DNA Super-Helicoidal , Óperon Lac , Autorradiografia , RNA Polimerases Dirigidas por DNA , Escherichia coli/genética , Regulação da Expressão Gênica , Plasmídeos , RNA Mensageiro , Moldes Genéticos , Transcrição GênicaRESUMO
The supercoiling response of four closely related promoters was examined in vitro. It was found that changes in all three elements of the lac ps promoter, i.e. the -10 sequence, the -35 sequence, and the spacer length, alter the transcriptional response to DNA supercoiling. Thus, the promoter as a whole, not just the melted region, mediates the supercoiling response. It is proposed that DNA supercoiling changes the structure of the promoter DNA to a form that can be recognized by RNA polymerase and then easily melted. All four promoter variants tested show the same qualitative response to the introduction of DNA supercoiling; that is, transcription is increased compared to relaxed DNA. However, for three of the four promoters, the rate peaked at intermediate levels of supercoiling and declined at higher superhelicities. Each mutation was found to alter both the extent of stimulation that can be achieved and the amount of superhelicity associated with maximal stimulation. The trend is that the stronger promoters are stimulated less, and this maximal stimulation occurs on templates containing fewer superhelical turns. At the level of supercoiling that may pertain in vivo, changes in superhelicity would result in considerable differential changes in the strengths of these promoters.
Assuntos
DNA Bacteriano , DNA Super-Helicoidal , Óperon Lac , Regiões Promotoras Genéticas , Transcrição Gênica , Escherichia coli/genética , MutaçãoRESUMO
Titration experiments on supercoiled lac DNA show that one repressor tetramer can bind simultaneously to the primary lac operator and to the very weak lac pseudo-operator, located 93 base-pairs apart. The formation of this complex is accompanied by the appearance of an extreme hypersensitive site in a five base-pair sequence located approximately midway between the operators. This remote sequence is hypersensitive to attack by two different chemical probes, dimethyl sulfate and potassium permanganate, the latter of which is a new probe for distorted DNA. We interpret these results in terms of a complex in which lac repressor holds two remote operators together in a DNA loop. The formation of this bent DNA loop requires negative DNA supercoiling. In vivo, both lac operators bind repressor even though the presence of multiple operator copies has forced the two operators to compete for a limited amount of repressor. This suggests that the operator and pseudo-operator have similar affinities for repressor in vivo. Such similar affinities were observed in vitro only when DNA supercoiling forced formation of a repression loop.
Assuntos
DNA Bacteriano , DNA Super-Helicoidal , Óperon Lac , Conformação de Ácido Nucleico , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Plasmídeos , Proteínas Repressoras/metabolismoRESUMO
Mutation of the simian virus 40 (SV40) origin of replication (ori) has revealed the presence of three critical domains needed for DNA replication. The outer two domains, the AT tract and early palindrome element (EP), colocalize with DNA regions that become structurally altered in the presence of the SV40 large tumor antigen (T antigen) and ATP. Mutations within each domain were examined for their effect on the distortion of ori DNA by T antigen, as assayed by the sensitivity of DNA to KMnO4 oxidation. We have found that mutations in the AT tract that inhibit SV40 DNA replication also inhibit the distortion of the AT tract. Similarly, mutations in the EP inhibited the generation of structural changes in this element by T antigen. Although AT-tract mutations or mutations on the late side of ori affected structural changes only in the AT tract, certain EP mutations or mutations on the early side of ori also inhibited AT-tract distortion. Mutation of the flanking regions did not significantly affect either the affinity of T antigen for ori or the rate of binding to ori. We conclude from these results that the primary function of the flanking ori domains is to undergo structural changes required during the initiation of SV40 DNA replication. Moreover, our results suggest that the efficiency of replication initiation is significantly affected by the degree to which the flanking elements undergo a structural transition.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Replicação do DNA/genética , DNA Viral/genética , Vírus 40 dos Símios/fisiologia , Replicação Viral/genética , Sítios de Ligação , DNA Viral/metabolismo , Vírus 40 dos Símios/genéticaRESUMO
The simian virus 40 (SV40) large tumor antigen(T antigen) has been shown to induce the melting of 8 bp within the SV40 origin of replication. We found previously that a 'pseudo-origin' DNA molecule (PO-8) containing a central 8 nt single-stranded DNA (ssDNA) bubble was efficiently bound and denatured by human replication protein A (hRPA). To understand the mechanism by which hRPA denatures these pseudo-origin molecules, as well as the role that hRPA plays during the initiation of SV40 DNA replication, we characterized the key parameters for the pseudo-origin binding and denaturation reactions. The dissociation constant of hRPA binding to PO-8 was observed to be 7.7 x 10(-7) M, compared to 9.0 x 10(-8) M for binding to an identical length ssDNA under the same reaction conditions. The binding and denaturation of PO-8 occurred with different kinetics with the rate of binding determined to be approximately 4-fold greater than the rate of denaturation. Although hRPA binding to PO-8 was relatively temperature independent, an increase in incubation temperature from 4 to 37 degreesC stimulated denaturation nearly 4-fold. At 37 degreesC, denaturation occurred on approximately 1/3 of those substrate molecules bound by hRPA, showing that hRPA can bind the pseudo-origin substrate without causing its complete denaturation. Tests of other single-stranded DNA-binding proteins (SSBs) over a range of SSB concentrations revealed that the ability of the SSBs to bind the pseudo-origin substrate, rather than denature the substrate, correlated best with the known ability of these SSBs to support the T antigen-dependent SV40 origin-unwinding activity. Our data indicate that hRPA first binds the DNA substrate using a combination of contacts with the ssDNA bubble and duplex DNA flanks and then, on only a fraction of the bound substrate molecules, denatures the DNA substrate.
Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Antígenos Virais de Tumores , Replicação do DNA , DNA Viral/química , Humanos , Desnaturação Proteica , Proteína de Replicação A , Vírus 40 dos Símios , Especificidade por SubstratoRESUMO
Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires >/=40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5' junction on each strand, with 5 bp of duplex DNA and approximately 17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5' direction into the duplex region, while resulting in no significant changes to the 3' edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.
Assuntos
Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/metabolismo , Replicação do DNA/imunologia , Replicação do DNA/fisiologia , Vírus 40 dos Símios/imunologia , Vírus 40 dos Símios/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , DNA Helicases/metabolismo , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Dimerização , Técnicas In Vitro , Modelos Biológicos , Conformação ProteicaRESUMO
Simian virus 40 (SV40) large tumor antigen (T antigen) binds to two contiguous sites at the SV40 origin of replication. Of these two sites, I and II, only site II is critical for replication. We have studied the interaction between T antigen and these sites by two methods--nitrocellulose filter binding and DNase I protection. We show that T antigen binds with high occupancy to site I at 0 degrees C, 25 degrees C, and 37 degrees C but to site II only at 0 degrees C and 25 degrees C. At 37 degrees C, the temperature essential for the initiation of SV40 DNA replication in vitro, ATP is required for the interaction of T antigen and site II. ATP can be replaced efficiently by adenosine 5'-[beta,gamma-imido]triphosphate and ADP, suggesting that hydrolysis of the nucleotide is not essential for the binding of T antigen to site II. The binding to the region critical for replication can occur in the presence of a variety of nucleoside triphosphates; dATP supports binding at a concentration 1/30th that of ATP, while dGTP and rGTP were inactive at all concentrations tested.
Assuntos
Trifosfato de Adenosina/farmacologia , Antígenos Transformantes de Poliomavirus , Replicação do DNA/efeitos dos fármacos , DNA Viral/genética , Vírus 40 dos Símios/genética , Nucleotídeos de Adenina/farmacologia , Animais , Linhagem Celular , DNA Viral/efeitos dos fármacos , Desoxirribonucleotídeos/farmacologia , Genes Virais , Cinética , Vírus 40 dos Símios/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Replication of simian virus 40 (SV40) DNA is dependent upon the binding of the viral T-antigen to the SV40 origin of replication. Structural changes in the origin of replication induced by binding of T-antigen were probed by chemical modifications of the DNA. In the presence of ATP, T-antigen rendered two of three domains in the SV40 core origin hypersensitive to attack by either dimethyl sulfate or potassium permanganate (KMnO4). One of these domains, the early palindrome, was shown to contain an 8-bp region of melted DNA as determined from methylation of cytosine residues and by nuclease S1 cleavage of methylated DNA. DNA melting was not dependent upon either the hydrolysis of ATP or the binding of T-antigen to an adjacent site (site I). A second domain, the A/T element, was extensively modified by KMnO4 but no significant melting was detected. Rather, the pattern of modification indicates that T-antigen caused a conformational change of the double-stranded DNA in this region. These results suggest that T-antigen, in the presence of ATP, destabilizes the SV40 origin by melting and structurally deforming two flanking regions within the core origin sequence. These DNA structural changes may provide access to other replication factors, allowing complete denaturation of the SV40 origin and the initiation of SV40 DNA replication.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Replicação do DNA , Proteínas de Ligação a DNA/fisiologia , Sequências Reguladoras de Ácido Nucleico , Vírus 40 dos Símios/genética , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Endonucleases/metabolismo , Metilação , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oxirredução , Permanganato de Potássio , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
The mechanism by which a replicator (origin of replication) becomes denatured during the initiation of replication is not understood for any prokaryotic or eukaryotic system. To address this question, we chemically probed the molecular contacts on the SV40 origin of replication (ori) that are used by the SV40 large T-antigen and a single-stranded DNA-binding protein (SSB) during ori denaturation. Prior to the actual denaturation step, the T-antigen double hexamer bound ori utilizing sugar-phosphate contacts that were located on opposite strands in each flanking domain of ori. Each set of flanking phosphate contacts were also located on approximately opposite faces of the ori duplex. While the phosphate contacts had a 2-fold symmetry with respect to the ori center, T-antigen contacts with nucleotide bases were polar with critical interactions detected in only one of the two flanking domains. During origin denaturation catalyzed by T-antigen and a SSB, numerous new contacts to flanking phosphates were observed on the strand not initially bound by T-antigen, suggesting movement of each T-antigen hexamer outward from ori. These data suggest that T-antigen initially binds ori in a manner that facilitates transfer of each T-antigen hexamer to opposite strands during the initiation of SV40 replication.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Replicação do DNA , DNA Viral/biossíntese , Vírus 40 dos Símios/genética , Animais , Baculoviridae/genética , Sequência de Bases , Linhagem Celular , DNA de Cadeia Simples/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Mariposas , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Fosfatos/metabolismo , Ligação Proteica , RepliconRESUMO
A new primer extension analysis is used to determine the methylation pattern over the lac UV5 promoter when dimethyl sulfate is added to growing Escherichia coli. The high-resolution analysis reveals altered methylation of 15 bases when the transcription machinery occupies the promoter inside the cell and shows a striking dichotomy in the distribution of methylated bases. Four protected guanosines lie on the side of the helix shown previously to be closely bound by RNA polymerase in vitro [Siebenlist, U., Simpson, R. B., & Gilbert, W. (1980) Cell (Cambridge, Mass.) 20, 269-281]. By contrast, the 11 hyperreactive bases lie on the side of the DNA directly opposite from that bound by protein. Those not in the melted region form two distinct "back-side" patches near -35 and -16. We suggest that such hyperreactive patches can be caused by proteins bending the DNA toward themselves to allow a full range of contacts, thus distorting the helix grooves on the "back" side and facilitating attack by the methylating reagent. This leads to a proposal for the formation of transcription complexes in which RNA polymerase interacts with deformed and torsionally stressed DNA.
Assuntos
Escherichia coli/genética , Óperon Lac , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Metilação , PlasmídeosRESUMO
Chromosomal DNA replication in higher eukaryotes takes place in DNA synthesis factories containing numerous replication forks. We explored the role of replication fork aggregation in vitro, using as a model the simian virus 40 (SV40) large tumor antigen (T antigen), essential for its DNA helicase and origin-binding activities. Previous studies have shown that T antigen binds model DNA replication forks primarily as a hexamer (TAgH) and to a lesser extent as a double hexamer (TAgDH). We find that DNA unwinding in the presence of ATP or other nucleotides strongly correlates with the formation of TAgDH-DNA fork complexes. TAgH- and TAgDH-fork complexes were isolated, and the TAgDH-bound fork was denatured at a 15-fold-higher rate during the initial times of unwinding. TAgDH bound preferentially to a DNA substrate containing a 50-nucleotide bubble, indicating the bridging of each single-stranded DNA/duplex DNA junction, and this DNA molecule was also unwound at a high rate. Both the TAgH- and TAgDH-fork complexes were relatively stable, with the half-life of the TAgDH-fork complex greater than 40 min. Our data therefore indicate that the linking of two viral replication forks serves to activate DNA replication.
Assuntos
Antígenos Transformantes de Poliomavirus/química , DNA Helicases/química , DNA Helicases/metabolismo , Replicação do DNA , Vírus 40 dos Símios/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Sistema Livre de Células , DNA Viral/biossíntese , Dimerização , Cinética , Proteínas Recombinantes , Vírus 40 dos Símios/enzimologia , Relação Estrutura-AtividadeRESUMO
Human replication protein A (hRPA) was previously seen to efficiently bind a 48 bp simian virus 40 (SV40) "pseudo-origin" (PO) substrate that mimics a DNA structure found within the SV40 T antigen-origin (ori) complex. To understand the role of hRPA during the initiation of replication, we examined the PO sequence and structure requirements for hRPA interaction. Binding and unwinding were found to be most efficient when both strands of the central 8 nt single-stranded DNA (ssDNA) bubble region contained a polypyrimidine structure, with these activities proportionately reduced when the bubble region was replaced with a purine tract on one or both strands. Examination of the importance of the two duplex flanks indicates that the early gene side contains a DNA structural feature located one duplex turn from the bubble whose mutation significantly affects the affinity of hRPA for the substrate. When present in the context of ori, mutation of this sequence was seen to have significant effects on SV40 DNA replication in vitro and on the denaturation of ori, indicating that origin activity can be modulated by cis-acting elements which alter the hRPA binding affinity. Use of fork and overhang substrates containing 8 nt pyrimidine or purine arms demonstrates that hRPA binding to DNA involves a particular molecular polarity in which initial hRPA binding occurs on the 5' side of a ssDNA substrate, and then extends in the 3' direction to create a stably bound hRPA. These data have implications on the mechanism of the initiation of eukaryotic DNA replication as well as on the sites of nascent strand synthesis within the origin.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Regiões 5' não Traduzidas/metabolismo , Replicação do DNA , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Vírus 40 dos Símios/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Composição de Bases , Sequência de Bases , Replicação do DNA/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico/genética , Ligação Proteica/genética , Nucleotídeos de Pirimidina/genética , Nucleotídeos de Pirimidina/metabolismo , Origem de Replicação/genética , Proteína de Replicação ARESUMO
The modulation of DNA replication by transcription factors was examined by using bovine papillomavirus type 1 (BPV). BPV replication in vivo requires two viral proteins: E1, an origin-binding protein, and E2, a transcriptional transactivator. In the origin, E1 interacts with a central region flanked by two binding sites for E2 (BS11 and BS12), of which only BS12 has been reported to be essential for replication in vivo. Using chemical interference and electrophoretic mobility shift assays, we found that the binding of E2 to each site stimulates the formation of distinct E1-origin complexes. A high-mobility C1 complex is formed by using critical E2 contacts to BS12 and E1 contacts to the dyad symmetry element. In contrast, interaction of E2 with the BS11 element on the other origin flank promotes the formation of the lower-mobility C3 complex. C3 is a novel species that resembles C2, a previously identified complex that is replication active and formed by E1 alone. The binding of E1 greatly differs in the C1 and C3 complexes, with E1 in the C1 complex limited to the origin dyad symmetry region and E1 in the C3 complex encompassing the region from the proximal edge of BS11 through the distal edge of BS12. We found that the presence of both E2-binding sites is necessary for wild-type replication activity in vivo, as well as for maximal production of the C3 complex. These results show that in the normal viral context, BS11 and BS12 play separate but synergetic roles in the initiation of viral DNA replication that are dependent on their location within the origin. Our data suggest a model in which the binding of E2 to each site sequentially stimulates the formation of distinct E1-origin complexes, leading to the replication-competent complex.
Assuntos
Papillomavirus Bovino 1/genética , Replicação do DNA , Proteínas de Ligação a DNA/fisiologia , Transativadores/fisiologia , Proteínas Virais/fisiologia , Replicação Viral , Animais , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , SpodopteraRESUMO
The ATP-dependent binding of the simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication (ori) results in the structural distortion of two critical elements within flanking regions of ori and the untwisting of the DNA helix. We examined the effect of changes in temperature, ATP concentration, and other reaction parameters on the generation of these DNA structural changes. We found that induction of the two localized structural transitions were highly and differentially sensitive to reaction conditions. Significant distortion of the early palindrome element, shown previously to result from DNA melting, required low levels of ATP (10 to 30 microM) but temperatures above 25 degrees C. Distortion of the AT tract occurred at low temperatures (5 degrees C) but required relatively high concentrations of ATP (greater than 300 microM). Thus, T antigen can induce structural changes within one critical element of ori without generating significant structural distortion within the second element. The response of ori untwisting to reaction conditions generally increased in parallel with or fell intermediate between the inductions of localized structural transitions. We suggest that ori untwisting and localized structural distortions are interdependent consequences of T-antigen binding to ori. These results suggest a model for the structural events occurring during the initial steps of SV40 DNA replication.