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1.
J Phys Chem Lett ; 10(22): 7200-7207, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31693374

RESUMO

DNA compaction is essential to ensure the packaging of the genetic material in living cells and also plays a key role in the epigenetic regulation of gene expression. In both humans and bacteria, DNA packaging is achieved by specific well-conserved proteins. Here, by means of all-atom molecular dynamics simulations, including the determination of relevant free-energy profiles, we rationalize the molecular bases for this remarkable process in bacteria, illustrating the crucial role played by positively charged amino acids of a small histone-like protein. We also present compelling evidence that this histone-like protein alone can induce strong bending of a DNA duplex around its core domain, a process that requires overcoming a major free-energy barrier.


Assuntos
Proteínas de Bactérias/química , Borrelia burgdorferi/química , Empacotamento do DNA , DNA Bacteriano/química , Histonas/química , Simulação de Dinâmica Molecular , Modelos Moleculares
2.
Nat Commun ; 10(1): 3815, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444361

RESUMO

Our knowledge of bacterial nucleoids originates mostly from studies of rod- or crescent-shaped bacteria. Here we reveal that Deinococcus radiodurans, a relatively large spherical bacterium with a multipartite genome, constitutes a valuable system for the study of the nucleoid in cocci. Using advanced microscopy, we show that D. radiodurans undergoes coordinated morphological changes at both the cellular and nucleoid level as it progresses through its cell cycle. The nucleoid is highly condensed, but also surprisingly dynamic, adopting multiple configurations and presenting an unusual arrangement in which oriC loci are radially distributed around clustered ter sites maintained at the cell centre. Single-particle tracking and fluorescence recovery after photobleaching studies of the histone-like HU protein suggest that its loose binding to DNA may contribute to this remarkable plasticity. These findings demonstrate that nucleoid organization is complex and tightly coupled to cell cycle progression in this organism.


Assuntos
Proteínas de Bactérias/metabolismo , Divisão Celular , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Deinococcus/fisiologia , Organelas/metabolismo , Ciclo Celular , DNA Bacteriano/genética , Loci Gênicos/fisiologia , Genoma Bacteriano/fisiologia , Microscopia Intravital , Microscopia de Fluorescência , Organelas/genética
3.
DNA Repair (Amst) ; 78: 45-59, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959406

RESUMO

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase that removes oxidized pyrimidines from DNA. The genome of Deinococcus radiodurans encodes for an unusually high number of DNA glycosylases, including three EndoIII enzymes (drEndoIII1-3). Here, we compare the properties of these enzymes to those of their well-studied homologues from E. coli and human. Our biochemical and mutational data, reinforced by MD simulations of EndoIII-DNA complexes, reveal that drEndoIII2 exhibits a broad substrate specificity and a catalytic efficiency surpassing that of its counterparts. In contrast, drEndoIII1 has much weaker and uncoupled DNA glycosylase and AP-lyase activities, a characteristic feature of eukaryotic DNA glycosylases, and was found to present a relatively robust activity on single-stranded DNA substrates. To our knowledge, this is the first report of such an activity for an EndoIII. In the case of drEndoIII3, no catalytic activity could be detected, but its ability to specifically recognize lesion-containing DNA using a largely rearranged substrate binding pocket suggests that it may play an alternative role in genome maintenance. Overall, these findings reveal that D. radiodurans possesses a unique set of DNA repair enzymes, including three non-redundant EndoIII variants with distinct properties and complementary activities, which together contribute to genome maintenance in this bacterium.


Assuntos
Reparo do DNA , DNA Complementar/genética , Deinococcus/enzimologia , Deinococcus/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Mutação , Biocatálise , DNA Complementar/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Pirimidinas/metabolismo , Especificidade por Substrato
4.
Nat Commun ; 10(1): 31, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30604765

RESUMO

DnaB helicases are motor proteins that couple ATP-hydrolysis to the loading of the protein onto DNA at the replication fork and to translocation along DNA to separate double-stranded DNA into single strands during replication. Using a network of conformational states, arrested by nucleotide mimics, we herein characterize the reaction coordinates for ATP hydrolysis, DNA loading and DNA translocation using solid-state NMR spectroscopy. AMP-PCP is used as pre-hydrolytic, ADP:AlF4- as transition state, and ADP as post-hydrolytic ATP mimic. 31P and 13C NMR spectra reveal conformational and dynamic responses to ATP hydrolysis and the resulting DNA loading and translocation with single amino-acid resolution. This allows us to identify residues guiding the DNA translocation process and to explain the high binding affinities for DNA observed for ADP:AlF4-, which turns out to be optimally preconfigured to bind DNA.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , DNA de Cadeia Simples/metabolismo , DnaB Helicases/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Replicação do DNA/fisiologia , DnaB Helicases/metabolismo , Hidrólise , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
5.
Sci Rep ; 8(1): 14038, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30232348

RESUMO

Spurious blinking fluorescent spots are often seen in bacteria during single-molecule localization microscopy experiments. Although this 'autoblinking' phenomenon is widespread, its origin remains unclear. In Deinococcus strains, we observed particularly strong autoblinking at the periphery of the bacteria, facilitating its comprehensive characterization. A systematic evaluation of the contributions of different components of the sample environment to autoblinking levels and the in-depth analysis of the photophysical properties of autoblinking molecules indicate that the phenomenon results from transient binding of fluorophores originating mostly from the growth medium to the bacterial cell wall, which produces single-molecule fluorescence through a Point Accumulation for Imaging in Nanoscale Topography (PAINT) mechanism. Our data suggest that the autoblinking molecules preferentially bind to the plasma membrane of bacterial cells. Autoblinking microscopy was used to acquire nanoscale images of live, unlabeled D. radiodurans and could be combined with PALM imaging of PAmCherry-labeled bacteria in two-color experiments. Autoblinking-based super-resolved images provided insight into the formation of septa in dividing bacteria and revealed heterogeneities in the distribution and dynamics of autoblinking molecules within the cell wall.


Assuntos
Parede Celular/ultraestrutura , Deinococcus/ultraestrutura , Imagem Individual de Molécula/métodos , Microscopia de Fluorescência , Nanotecnologia/métodos
7.
Angew Chem Int Ed Engl ; 55(45): 14164-14168, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27709753

RESUMO

DnaB helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues.


Assuntos
DNA de Cadeia Simples/metabolismo , DnaB Helicases/metabolismo , Helicobacter pylori/metabolismo , Ressonância Magnética Nuclear Biomolecular , Sítios de Ligação , DNA de Cadeia Simples/análise , DnaB Helicases/química , Helicobacter pylori/química
8.
Nucleic Acids Res ; 43(17): 8564-76, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26264665

RESUMO

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.


Assuntos
Proteínas de Bactérias/química , DNA Primase/metabolismo , DnaB Helicases/química , Helicobacter pylori/enzimologia , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/metabolismo , DNA Primase/química , DNA de Cadeia Simples/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Ativação Enzimática , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína
9.
J Struct Biol ; 191(2): 87-99, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26172070

RESUMO

While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity.


Assuntos
Proteínas de Bactérias/química , Deinococcus/enzimologia , Desoxirribonuclease (Dímero de Pirimidina)/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Clonagem Molecular , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de Proteína , Relação Estrutura-Atividade
10.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 12): 1688-92, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25484227

RESUMO

Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Šresolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, ß = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, ß = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.


Assuntos
Deinococcus/enzimologia , Endonucleases/metabolismo , Cristalização , Cristalografia por Raios X , Endonucleases/química
11.
PLoS One ; 8(10): e77364, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24143224

RESUMO

DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3'-5' direction and shows ATP-dependent 3'-5', and surprisingly also, 5'-3' helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved ß-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD's 3'-5' and 5'-3' helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5'-3' helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo.


Assuntos
DNA Helicases/metabolismo , DNA/química , DNA/metabolismo , Deinococcus/enzimologia , Conformação de Ácido Nucleico , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Cristalografia por Raios X , DNA Helicases/química , DNA Helicases/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Mutagênese , Conformação Proteica
12.
Nucleic Acids Res ; 41(16): 7972-86, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23814185

RESUMO

Efficient DNA repair is critical for cell survival and the maintenance of genome integrity. The homologous recombination pathway is responsible for the repair of DNA double-strand breaks within cells. Initiation of this pathway in bacteria can be carried out by either the RecBCD or the RecFOR proteins. An important regulatory player within the RecFOR pathway is the RecOR complex that facilitates RecA loading onto DNA. Here we report new data regarding the assembly of Deinococcus radiodurans RecOR and its interaction with DNA, providing novel mechanistic insight into the mode of action of RecOR in homologous recombination. We present a higher resolution crystal structure of RecOR in an 'open' conformation in which the tetrameric RecR ring flanked by two RecO molecules is accessible for DNA binding. We show using small-angle neutron scattering and mutagenesis studies that DNA binding does indeed occur within the RecR ring. Binding of single-stranded DNA occurs without any major conformational changes of the RecOR complex while structural rearrangements are observed on double-stranded DNA binding. Finally, our molecular dynamics simulations, supported by our biochemical data, provide a detailed picture of the DNA binding motif of RecOR and reveal that single-stranded DNA is sandwiched between the two facing oligonucleotide binding domains of RecO within the RecR ring.


Assuntos
Proteínas de Bactérias/química , DNA de Cadeia Simples/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Deinococcus , Modelos Moleculares , Mutagênese , Conformação Proteica
13.
Structure ; 20(12): 2076-89, 2012 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-23085075

RESUMO

Repair of DNA double-strand breaks (DSBs) is essential for cell survival and maintaining genome integrity. DSBs are repaired in a stepwise manner by homologous recombination. Here, we focused on the early steps of DSB repair, including DSB recognition, which is still only poorly understood. In prokaryotes, this process has been proposed to involve the RecN protein, a member of the structural maintenance of chromosome (SMC) protein family, which include key eukaryotic and prokaryotic proteins such as cohesin, condensin, and Rad50. An extensive high- and low-resolution structural analysis of Deinococcus radiodurans RecN using a combination of protein crystallography and small-angle X-ray scattering enabled us to assemble a quasi-atomic model of the entire RecN protein, representing the complete structure of a SMC-like protein. These results, together with a thorough biochemical and mutational study of RecN, allow us to propose a model for the role of RecN in DSB repair.


Assuntos
Proteínas de Bactérias/química , Enzimas de Restrição do DNA/química , Deinococcus , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas de Restrição do DNA/genética , Ligações de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo
14.
Plant Physiol ; 160(1): 249-60, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22822210

RESUMO

Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.


Assuntos
Ácido Clorogênico/química , Café/química , Ácido Quínico/análogos & derivados , Aciltransferases/química , Aciltransferases/genética , Sequência de Aminoácidos , Aminoácidos/química , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Coffea/química , Coffea/genética , Ativação Enzimática , Escherichia coli/química , Escherichia coli/genética , Ésteres/química , Isomerismo , Conformação Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Biossíntese de Proteínas , Ácido Quínico/química , Sementes/química , Sementes/genética , Alinhamento de Sequência , Especificidade por Substrato
15.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 6): 703-12, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22683793

RESUMO

3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/ß domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1,N(6)-ethenoadenine (ℇA) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket.


Assuntos
DNA Glicosilases/química , Deinococcus/enzimologia , Sequência de Aminoácidos , DNA/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Especificidade por Substrato
16.
Structure ; 20(3): 554-64, 2012 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-22405014

RESUMO

Hexameric DnaB helicases are often loaded at DNA replication forks by interacting with the initiator protein DnaA and/or a helicase loader (DnaC in Escherichia coli). These loaders are not universally required, and DnaB from Helicobacter pylori was found to bypass DnaC when expressed in E. coli cells. The crystal structure of Helicobacter pylori DnaB C-terminal domain (HpDnaB-CTD) reveals a large two-helix insertion (named HPI) in the ATPase domain that protrudes away from the RecA fold. Biophysical characterization and electron microscopy (EM) analysis of the full-length protein show that HpDnaB forms head-to-head double hexamers remarkably similar to helicases found in some eukaryotes, archaea, and viruses. The docking of the HpDnaB-CTD structure into EM reconstruction of HpDnaB provides a model that shows how hexamerization of the CTD is facilitated by HPI-HPI interactions. The HpDnaB double-hexamer architecture supports an alternative strategy to load bacterial helicases onto forks in the absence of helicase loaders.


Assuntos
DnaB Helicases/química , Helicobacter pylori/enzimologia , Modelos Moleculares , Conformação Proteica , Escherichia coli , Microscopia Eletrônica , Polimerização , Ligação Proteica , Origem de Replicação/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-22298004

RESUMO

Deinococcus radiodurans has developed an efficient mechanism which allows the integrity of its entire genome to be fully restored after exposure to very high doses of ionizing radiation. Homologous recombination plays a crucial role in this process. RecN is a protein that belongs to the SMC-like protein family and is suggested to be involved in DNA repair. RecN is composed of a globular domain and an antiparallel coiled-coil region which connects the N- and C-termini. It has been suggested that dimerization of RecN occurs via the coiled-coil domain, but to date there is no structural or biochemical evidence for this. Here, SAXS studies and preliminary X-ray diffraction data of crystals of the purified coiled-coil domain of RecN are presented. The structure was solved by single-wavelength anomalous dispersion using SeMet derivatives, and preliminary electron-density maps support the rod-like model derived from the SAXS data. Model building and refinement are still ongoing.


Assuntos
Proteínas de Bactérias/química , Enzimas de Restrição do DNA/química , Deinococcus/enzimologia , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Enzimas de Restrição do DNA/isolamento & purificação , Expressão Gênica , Modelos Moleculares , Estrutura Terciária de Proteína
18.
Artigo em Inglês | MEDLINE | ID: mdl-22232179

RESUMO

Deinococcus radiodurans is well known for its extreme tolerance to harsh conditions and for its extraordinary ability to repair DNA. Double-strand breaks (DSBs) are the most hazardous lesions that can be induced by ionizing radiation, and homologous recombination (HR) is the principal mechanism by which the integrity of the DNA is restored. In D. radiodurans the RecFOR complex is the main actor in HR and the RecN protein is believed to play an important role in DSB recognition. Here, SAXS and preliminary X-ray diffraction studies are presented of the head domain, which is the globular region formed upon interaction of the N- and C-terminal domains of RecN. The crystal structure of this domain was solved using the single-wavelength anomalous dispersion method. Model building and refinement are in progress.


Assuntos
Proteínas de Bactérias/química , Enzimas de Restrição do DNA/química , Deinococcus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/isolamento & purificação , Expressão Gênica , Modelos Moleculares , Estrutura Terciária de Proteína
19.
Structure ; 17(4): 547-58, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19368888

RESUMO

UvrA proteins are key actors in DNA damage repair and play an essential role in prokaryotic nucleotide excision repair (NER), a pathway that is unique in its ability to remove a broad spectrum of DNA lesions. Understanding the DNA binding and damage recognition activities of the UvrA family is a critical component for establishing the molecular basis of this process. Here we report the structure of the class II UvrA2 from Deinococcus radiodurans in two crystal forms. These structures, coupled with mutational analyses and comparison with the crystal structure of class I UvrA from Bacillus stearothermophilus, suggest a previously unsuspected role for the identified insertion domains of UvrAs in both DNA binding and damage recognition. Taken together, the available information suggests a model for how UvrA interacts with DNA and thus sheds new light on the molecular mechanisms underlying the role of UvrA in the early steps of NER.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Dano ao DNA , DNA Bacteriano/metabolismo , Deinococcus/enzimologia , Adenosina Trifosfatases/classificação , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalização , Análise Mutacional de DNA , Reparo do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Geobacillus stearothermophilus/enzimologia , Hidrólise , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Temperatura Ambiente , Fatores de Tempo
20.
Phys Rev Lett ; 103(19): 198102, 2009 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-20365956

RESUMO

X-ray diffraction microscopy (XDM) is well suited for nondestructive, high-resolution biological imaging, especially for thick samples, with the high penetration power of x rays and without limitations imposed by a lens. We developed nonvacuum, cryogenic (cryo-) XDM with hard x rays at 8 keV and report the first frozen-hydrated imaging by XDM. By preserving samples in amorphous ice, the risk of artifacts associated with dehydration or chemical fixation is avoided, ensuring the imaging condition closest to their natural state. The reconstruction shows internal structures of intact D. radiodurans bacteria in their natural contrast.


Assuntos
Deinococcus/citologia , Congelamento , Microscopia/métodos , Difração de Raios X/métodos , Cristalização , Deinococcus/ultraestrutura , Água/metabolismo
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