RESUMO
Inborn errors of DNA repair or replication underlie a variety of clinical phenotypes. We studied 5 patients from 4 kindreds, all of whom displayed intrauterine growth retardation, chronic neutropenia, and NK cell deficiency. Four of the 5 patients also had postnatal growth retardation. The association of neutropenia and NK cell deficiency, which is unusual among primary immunodeficiencies and bone marrow failures, was due to a blockade in the bone marrow and was mildly symptomatic. We discovered compound heterozygous rare mutations in Go-Ichi-Ni-San (GINS) complex subunit 1 (GINS1, also known as PSF1) in the 5 patients. The GINS complex is essential for eukaryotic DNA replication, and homozygous null mutations of GINS component-encoding genes are embryonic lethal in mice. The patients' fibroblasts displayed impaired GINS complex assembly, basal replication stress, impaired checkpoint signaling, defective cell cycle control, and genomic instability, which was rescued by WT GINS1. The residual levels of GINS1 activity reached 3% to 16% in patients' cells, depending on their GINS1 genotype, and correlated with the severity of growth retardation and the in vitro cellular phenotype. The levels of GINS1 activity did not influence the immunological phenotype, which was uniform. Autosomal recessive, partial GINS1 deficiency impairs DNA replication and underlies intra-uterine (and postnatal) growth retardation, chronic neutropenia, and NK cell deficiency.
Assuntos
Proteínas de Ligação a DNA/deficiência , Doenças Genéticas Inatas , Transtornos do Crescimento , Síndromes de Imunodeficiência , Células Matadoras Naturais , Neutropenia , Animais , Proteínas de Ligação a DNA/imunologia , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/imunologia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/imunologia , Transtornos do Crescimento/genética , Transtornos do Crescimento/imunologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Lactente , Masculino , Camundongos , Neutropenia/genética , Neutropenia/imunologiaRESUMO
Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.
Assuntos
Ligases/antagonistas & inibidores , Suramina/farmacologia , Relação Estrutura-AtividadeRESUMO
Despite minimal disparity at the sequence level, mammalian H3 variants bind to distinct sets of polypeptides. Although histone H3.1 predominates in cycling cells, our knowledge of the soluble complexes that it forms en route to deposition or following eviction from chromatin remains limited. Here, we provide a comprehensive analysis of the H3.1-binding proteome, with emphasis on its interactions with histone chaperones and components of the replication fork. Quantitative mass spectrometry revealed 170 protein interactions, whereas a large-scale biochemical fractionation of H3.1 and associated enzymatic activities uncovered over twenty stable protein complexes in dividing human cells. The sNASP and ASF1 chaperones play pivotal roles in the processing of soluble histones but do not associate with the active CDC45/MCM2-7/GINS (CMG) replicative helicase. We also find TONSL-MMS22L to function as a H3-H4 histone chaperone. It associates with the regulatory MCM5 subunit of the replicative helicase.
Assuntos
Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Espectrometria de Massas/métodos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Ligação ProteicaRESUMO
Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , RecQ Helicases/fisiologia , Origem de Replicação , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Replicação do DNA , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Emerging evidence suggests that Cdc13-Stn1-Ten1 (CST), an RPA-like ssDNA-binding complex, may regulate primase-Pol α (PP) activity at telomeres constitutively, and at other genomic locations under conditions of replication stress. Here we examine the mechanisms of PP stimulation by CST using purified complexes derived from Candida glabrata. While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching. CST also simultaneously shortens the RNA and lengthens the DNA in the chimeric products. Stn1, the most conserved subunit of CST, is alone capable of PP stimulation. Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity. Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.
Assuntos
Candida glabrata/genética , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Proteínas de Ligação a Telômeros/química , Telômero/química , Sítios de Ligação , Candida glabrata/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase I/genética , DNA Primase/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Instabilidade Genômica , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Fúngico/genética , RNA Fúngico/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(ßTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.
Assuntos
Lisina/metabolismo , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Substituição de Aminoácidos , Apoptose/genética , Biocatálise , Linhagem Celular Tumoral , Células HEK293 , Humanos , Ligação de Hidrogênio , Immunoblotting , Lisina/química , Lisina/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Poliubiquitina/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/química , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA.
Assuntos
Proteínas Arqueais/química , Antígeno Nuclear de Célula em Proliferação/química , Substituição de Aminoácidos , Proteínas Arqueais/genética , DNA Polimerase II/química , Medição da Troca de Deutério , Endonucleases Flap/química , Cinética , Viabilidade Microbiana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Thermococcus/fisiologiaRESUMO
Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT(Δ69-83)), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.
Assuntos
Antígenos Virais de Tumores/metabolismo , Proteínas Culina/antagonistas & inibidores , Proteínas Substratos do Receptor de Insulina/metabolismo , Transdução de Sinais/fisiologia , Vírus 40 dos Símios/química , Análise de Variância , Animais , Proteínas Culina/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Microscopia , Microscopia de Fluorescência , Proteólise , Interferência de RNA , Vírus 40 dos Símios/metabolismo , Ubiquitina/metabolismoRESUMO
Chromosome transmission fidelity 4 (Ctf4) is a conserved protein required for DNA replication. In this report, interactions between human Ctf4 (hCtf4) and the replicative helicase containing the cell division cycle 45 (Cdc45)/minichromosome maintenance 2-7 (Mcm2-7)/Go, Ichi, Nii, and San (GINS) (CMG) proteins [human CMG (hCMG) complex] were examined. The hCtf4-CMG complex was isolated following in vitro interaction of purified proteins (hCtf4 plus the hCMG complex), coinfection of Spodoptera frugiperda (Sf9) insect cells with viruses expressing the hCMG complex and hCtf4, and from HeLa cell chromatin after benzonase and immunoprecipitation steps. The stability of the hCtf4-CMG complex depends upon interactions between hCtf4 and multiple components of the hCMG complex. The hCtf4-CMG complex, like the hCMG complex, contains DNA helicase activity that is more salt-resistant than the helicase activity of the hCMG complex. We demonstrate that the hCtf4-CMG complex contains a homodimeric hCtf4 and a monomeric hCMG complex and suggest that the homodimeric hCtf4 acts as a platform linking polymerase α to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Primers do DNA/genética , Densitometria , Dimerização , Humanos , Imunoprecipitação , Proteínas de Manutenção de Minicromossomo/metabolismo , Oligonucleotídeos/genética , Células Sf9 , SpodopteraRESUMO
The repair of DNA breaks by homologous recombination is a high-fidelity process, necessary for the maintenance of genome integrity. Thus, DNA synthesis associated with recombinational repair must be largely error-free. In this report, we show that human DNA polymerase delta (δ) is capable of robust DNA synthesis at RAD51-mediated recombination intermediates dependent on the processivity clamp PCNA. Translesion synthesis polymerase eta (η) also extends these substrates, albeit far less processively. The single-stranded DNA binding protein RPA facilitates recombination-mediated DNA synthesis by increasing the efficiency of primer utilization, preventing polymerase stalling at specific sequence contexts, and overcoming polymerase stalling caused by topological constraint allowing the transition to a migrating D-loop. Our results support a model whereby the high-fidelity replicative DNA polymerase δ performs recombination-associated DNA synthesis, with translesion synthesis polymerases providing a supportive role as in normal replication.
Assuntos
DNA Polimerase III/metabolismo , DNA/biossíntese , Reparo de DNA por Recombinação , Proteína de Replicação A/metabolismo , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Rad51 Recombinase/metabolismoRESUMO
Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively. Here, we establish that both PCNA1 and PCNA2 form homotrimers that stimulate DNA synthesis by archaeal DNA polymerases B and D and ATP hydrolysis by the replication factor C complex. In exponentially growing cells, PCNA1 is abundant and present at an ~100-fold higher concentration than PCNA2 monomers. Deletion of TK0582 (PCNA2) had no detectable effects on viability or growth whereas repeated attempts to construct a T. kodakarensis strain with TK0535 (PCNA1) deleted were unsuccessful. The implications of these observations for PCNA1 function and the origin of the two PCNA-encoding genes in T. kodakarensis are discussed.
Assuntos
Proteínas Arqueais/genética , Viabilidade Microbiana/genética , Antígeno Nuclear de Célula em Proliferação/genética , Thermococcus/genética , Proteínas Arqueais/metabolismo , Replicação do DNA , Deleção de Genes , Antígeno Nuclear de Célula em Proliferação/metabolismo , Multimerização Proteica , Thermococcus/metabolismoRESUMO
Tim (Timeless) and Tipin (Tim-interacting protein) form a stable heterodimeric complex that influences checkpoint responses and replication fork progression. We report that the Tim-Tipin complex interacts with essential replication fork proteins and affects their biochemical properties. The Tim-Tipin complex, reconstituted and purified using the baculovirus expression system, interacts directly with Mcm complexes and inhibits the single-stranded DNA-dependent ATPase activities of the Mcm2-7 and Mcm4/6/7 complexes, the DNA unwinding activity of the Mcm4/6/7 complex, and the DNA unwinding and ATPase activity of Cdc45-Mcm2-7-GINS complex, the presumed replicative DNA helicase in eukaryotes. Although stable interactions between Tim-Tipin and DNA polymerases (pols) were not observed in immunoprecipitation experiments with purified proteins, Tim was shown to interact with DNA pols α, δ, and ε in cells. Furthermore, the Tim-Tipin complex significantly stimulated the pol activities of DNA pols α, δ, and ε in vitro. The effects of Tim-Tipin on the catalytic activities of the Mcm complexes and DNA pols are mediated by the Tim protein alone, and distinct regions of the Tim protein are responsible for the inhibition of Mcm complex activities and stimulation of DNA pols. These results suggest that the Tim-Tipin complex might play a role in coupling DNA unwinding and DNA synthesis by directly affecting the catalytic activities of replication fork proteins.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a DNA , Células HeLa , Humanos , Imunoprecipitação , Oligonucleotídeos/genéticaRESUMO
Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (known as steric exclusion). By contrast, large T antigen, the replicative DNA helicase of the simian virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here we use single-molecule and ensemble assays to show that large T antigen assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3'-to-5' direction. Unexpectedly, large T antigen unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the large T antigen ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative helicase mechanisms, and reveal a new level of plasticity in the interactions of replicative helicases with DNA damage.
Assuntos
DNA Helicases/metabolismo , Vírus 40 dos Símios/enzimologia , Antígenos Virais de Tumores/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Origem de Replicação/fisiologia , Proteínas Virais/metabolismoAssuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Circular/biossíntese , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , DNA Circular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo , Componente 7 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/metabolismoRESUMO
The loading of cohesin onto chromatin requires the heterodimeric complex sister chromatid cohesion (Scc)2 and Scc4 (Scc2/4), which is highly conserved in all species. Here, we describe the purification of the human (h)-Scc2/4 and show that it interacts with h-cohesin and the heterodimeric Smc1-Smc3 complex but not with the Smc1 or Smc3 subunit alone. We demonstrate that both h-Scc2/4 and h-cohesin are loaded onto dsDNA containing the prereplication complex (pre-RC) generated in vitro by Xenopus high-speed soluble extracts. The addition of geminin, which blocks pre-RC formation, prevents the loading of Scc2/4 and cohesin. Xenopus extracts depleted of endogenous Scc2/4 with specific antibodies, although able to form pre-RCs, did not support cohesin loading unless supplemented with purified h-Scc2/4. The results presented here indicate that the Xenopus or h-Scc2/4 complex supports the loading of Xenopus and/or h-cohesin onto pre-RCs formed by Xenopus high-speed extracts. We show that cohesin loaded onto pre-RCs either by h-Scc2/4 and/or the Xenopus complex was dissociated from chromatin by low salt extraction, similar to cohesin loaded onto chromatin in G(1) by HeLa cells in vivo. Replication of cohesin-loaded DNA, both in vitro and in vivo, markedly increased the stability of cohesin associated with DNA. Collectively, these in vitro findings partly recapitulate the in vivo pathway by which sister chromatids are linked together, leading to cohesion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Animais , Ciclo Celular , Cromatina/metabolismo , Dimerização , Humanos , Xenopus , CoesinasRESUMO
In eukaryotes, although the Mcm2-7 complex is a key component of the replicative DNA helicase, its association with Cdc45 and GINS (the CMG complex) is required for the activation of the DNA helicase. Here, we show that the CMG complex is localized to chromatin in human cells and describe the biochemical properties of the human CMG complex purified from baculovirus-infected Sf9 cells. The isolated complex binds to ssDNA regions in the presence of magnesium and ATP (or a nonhydrolyzable ATP analog), contains maximal DNA helicase in the presence of forked DNA structures, and translocates along the leading strand (3' to 5' direction). The complex hydrolyses ATP in the absence of DNA; unwinds duplex regions up to 500 bp; and either replication protein A or Escherichia coli single stranded binding protein increases the efficiency of displacement of long duplex regions. Using a 200-nt primed circular DNA substrate, the combined action of human DNA polymerase ε and the human CMG complex leads to the formation of products >10 kb in length. These findings suggest that the coordinated action of these replication complexes supports leading strand synthesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase II/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA/química , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Células HeLa , Humanos , Cinética , Componente 2 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Spodoptera , Especificidade por SubstratoRESUMO
In the presence of dATP, glycerol, and Tris buffer, the DNA primase isolated from Thermococcus kodakaraensis catalyzed the formation of dAMP and two products that were identified as dAMP-glycerol and dAMP-Tris. These products were formed by the T. kodakaraensis p41 catalytic subunit alone and the T. kodakaraensis p41-p46 complex in the absence of a DNA template. They were not formed with preparations containing the catalytically inactive p41 subunit. Similar glycerol and Tris derivatives as well as dNMPs were also formed with dGTP, dCTP, or dTTP. The mechanism contributing to the formation of these products and its implications in the initiation reaction catalyzed by the T. kodakaraensis primase are discussed.
Assuntos
DNA Primase/química , Nucleotídeos de Desoxiadenina/química , Glicerol/química , Thermococcus/enzimologia , Trometamina/química , Proteínas Arqueais/química , CatáliseRESUMO
In most organisms, DNA replication is initiated by DNA primases, which synthesize primers that are elongated by DNA polymerases. In this study, we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. kodakaraensis DNA primase complex is a heterodimer containing stoichiometric levels of the p41 and p46 subunits. The catalytic activity of the complex resides within the p41 subunit. We show that the complex supports both DNA and RNA synthesis, whereas the p41 subunit alone marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the T. kodakaraensis primase complex preferentially interacts with dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA chains de novo. The latter findings indicate that the archaeal primase complex, in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the absence of ribonucleoside triphosphates. DNA primers formed by the archaeal complex can be elongated extensively by the T. kodakaraensis DNA polymerase (Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. Supplementation of reactions containing the p41 subunit with the p46 subunit leads to PolB-catalyzed DNA synthesis. We also established a rolling circle reaction using a primed 200-nucleotide circle as the substrate. In the presence of the T. kodakaraensis minichromosome maintenance (MCM) 3' â 5' DNA helicase, PolB, replication factor C, and proliferating cell nuclear antigen, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.
Assuntos
Proteínas Arqueais/metabolismo , DNA Primase/metabolismo , Replicação do DNA/fisiologia , DNA Arqueal/biossíntese , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , Thermococcus/enzimologia , Proteínas Arqueais/genética , DNA Primase/genética , DNA Arqueal/genética , Complexos Multiproteicos/genética , Subunidades Proteicas/genética , RNA Arqueal/genética , RNA Arqueal/metabolismo , Thermococcus/genéticaRESUMO
The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3' to 5' ssDNA translocase, consistent with unwinding via "steric exclusion." Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA/metabolismo , Xenopus/metabolismo , Animais , DNA de Cadeia Simples/metabolismo , Modelos Biológicos , Fase SRESUMO
In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.