RESUMO
Divalent sulfur (S) forms a chalcogen bond (Ch-bond) via its σ-holes and a hydrogen bond (H-bond) via its lone pairs. The relevance of these interactions and their interplay for protein structure and function is unclear. Based on the analyses of the crystal structures of small organic/organometallic molecules and proteins and their molecular electrostatic surface potential, we show that the reciprocity of the substituent-dependent strength of the σ-holes and lone pairs correlates with the formation of either Ch-bond or H-bond. In proteins, cystines preferentially form Ch-bonds, metal-chelated cysteines form H-bonds, while methionines form either of them with comparable frequencies. This has implications for the positioning of these residues and their role in protein structure and function. Computational analyses reveal that the S-mediated interactions stabilise protein secondary structures by mechanisms such as helix capping and protecting free ß-sheet edges by negative design. The study highlights the importance of S-mediated Ch-bond and H-bond for understanding protein folding and function, the development of improved strategies for protein/peptide structure prediction and design and structure-based drug discovery.
RESUMO
Proteins and their assemblies are fundamental for living cells to function. Their complex three-dimensional architecture and its stability are attributed to the combined effect of various noncovalent interactions. It is critical to scrutinize these noncovalent interactions to understand their role in the energy landscape in folding, catalysis, and molecular recognition. This Review presents a comprehensive summary of unconventional noncovalent interactions, beyond conventional hydrogen bonds and hydrophobic interactions, which have gained prominence over the past decade. The noncovalent interactions discussed include low-barrier hydrogen bonds, C5 hydrogen bonds, C-H···π interactions, sulfur-mediated hydrogen bonds, n â π* interactions, London dispersion interactions, halogen bonds, chalcogen bonds, and tetrel bonds. This Review focuses on their chemical nature, interaction strength, and geometrical parameters obtained from X-ray crystallography, spectroscopy, bioinformatics, and computational chemistry. Also highlighted are their occurrence in proteins or their complexes and recent advances made toward understanding their role in biomolecular structure and function. Probing the chemical diversity of these interactions, we determined that the variable frequency of occurrence in proteins and the ability to synergize with one another are important not only for ab initio structure prediction but also to design proteins with new functionalities. A better understanding of these interactions will promote their utilization in designing and engineering ligands with potential therapeutic value.
RESUMO
The directionality of the chalcogen bond (Ch-bond) formed by S and its interplay with other weak interactions have important chemical and biological implications. Here, dimers made of CH3-S-X and O/N containing nucleophiles are studied and found to be stabilized by coexisting S···O/N and C-H···O/N interactions. Based on experimentally accessible electron density and molecular electrostatic potentials (MESPs), we showed that reciprocity between S···O/N and C-H···O/N interactions in the stability of cumulative molecular interaction (ΔE) was dependent on the strength of the σ-hole on S (Vs,max). Direct correlation between ΔE of dimers with Vs,max of S supports the electrostatic nature of the Ch-bond. Such interplay of the Ch-bond is necessary for its directionality in complex nucleophiles (carbonyl groups) with multiple electron-rich centers, which is explained using MESP. A correlation between the MESP minima in the π-region and the strength of the S-π interaction explains the directional selectivity of the Ch-bond.
Assuntos
Calcogênios , Elétrons , Calcogênios/química , Ligação de Hidrogênio , Eletricidade EstáticaRESUMO
Mechanisms that target and destroy foreign nucleic acids are major barriers to horizontal gene transfer (HGT) in prokaryotes. Amongst them, restriction-modification (R-M) systems are found in ≥75% of the sequenced genomes in Bacteria and Archaea. Due to their high target sequence specificity and potent nucleolytic activity, R-M systems are used as a paradigm to elucidate the mechanisms of DNA binding and cleavage. Since these enzymes modulate HGT, they are one of the machineries implicated in the ability of a bacterium to gain antibiotic resistance. This protocol provides a detailed purification strategy for the Type IV restriction endonuclease SauUSI from Staphylococcus aureus. This protocol eventually leads to ≥95% purity of protein which can then be used for crystallographic and biochemical purposes. Graphic abstract: Workflow for purification of SauUSI.
RESUMO
Acquisition of foreign DNA by Staphylococcus aureus, including vancomycin resistance genes, is thwarted by the ATP-dependent endonuclease SauUSI. Deciphering the mechanism of action of SauUSI could unravel the reason how it singularly plays a major role in preventing horizontal gene transfer (HGT) in S. aureus. Here, we report a detailed biochemical and structural characterization of SauUSI, which reveals that in the presence of ATP, the enzyme can cleave DNA having a single or multiple target site/s. Remarkably, in the case of multiple target sites, the entire region of DNA flanked by two target sites is shred into smaller fragments by SauUSI. Crystal structure of SauUSI reveals a stable dimer held together by the nuclease domains, which are spatially arranged to hydrolyze the phosphodiester bonds of both strands of the duplex. Thus, the architecture of the dimeric SauUSI facilitates cleavage of either single-site or multi-site DNA. The structure also provides insights into the molecular basis of target recognition by SauUSI. We show that target recognition activates ATP hydrolysis by the helicase-like ATPase domain, which powers active directional movement (translocation) of SauUSI along the DNA. We propose that a pile-up of multiple translocating SauUSI molecules against a stationary SauUSI bound to a target site catalyzes random double-stranded breaks causing shredding of the DNA between two target sites. The extensive and irreparable damage of the foreign DNA by shredding makes SauUSI a potent barrier against HGT.
Assuntos
Clivagem do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Staphylococcus aureus/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA/química , Farmacorresistência Bacteriana , Transferência Genética Horizontal , Modelos Moleculares , Multimerização Proteica , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
Enzymes involved in nucleic acid transactions often have a helicase-like ATPase coordinating and driving their functional activities, but our understanding of the mechanistic details of their coordination is limited. For example, DNA cleavage by the antiphage defense system Type ISP restriction-modification enzyme requires convergence of two such enzymes that are actively translocating on DNA powered by Superfamily 2 ATPases. The ATPase is activated when the enzyme recognizes a DNA target sequence. Here, we show that the activation is a two-stage process of partial ATPase stimulation upon recognition of the target sequence by the methyltransferase and the target recognition domains, and complete stimulation that additionally requires the DNA to interact with the ATPase domain. Mutagenesis revealed that a ß-hairpin loop and motif V of the ATPase couples DNA translocation to ATP hydrolysis. Deletion of the loop inhibited translocation, while mutation of motif V slowed the rate of translocation. Both the mutations inhibited the double-strand (ds) DNA cleavage activity of the enzyme. However, a translocating motif V mutant cleaved dsDNA on encountering a translocating wild-type enzyme. Based on these results, we conclude that the ATPase-driven translocation not only brings two nucleases spatially close to catalyze dsDNA break, but that the rate of translocation influences dsDNA cleavage.
Assuntos
Adenosina Trifosfatases/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Adenosina Trifosfatases/química , Motivos de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA/química , Ativação Enzimática , Mutação/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Deleção de Sequência , Especificidade por SubstratoRESUMO
S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) are involved in diverse cellular functions. These enzymes show little sequence conservation but have a conserved structural fold. The DNA MTases have characteristic motifs that are involved in AdoMet binding, DNA target recognition and catalysis. Motif III of these MTases have a highly conserved acidic residue, often an aspartate, whose functional significance is not clear. Here, we report a mutational study of the residue in the ß family MTase of the Type III restriction-modification enzyme EcoP15I. Replacement of this residue by alanine affects its methylation activity. We propose that this residue contributes to the affinity of the enzyme for AdoMet. Analysis of the structures of DNA, RNA and protein MTases reveal that the acidic residue is conserved in all of them, and interacts with N6 of the adenine moiety of AdoMet. Interestingly, in the SET-domain protein lysine MTases, which have a fold different from other AdoMet-dependent MTases, N6 of the adenine moiety is hydrogen bonded to the main chain carbonyl group of the histidine residue of the highly conserved motif III. Our study reveals the evolutionary conservation of a carbonyl group in DNA, RNA and protein AdoMet-dependent MTases for specific interaction by hydrogen bond with AdoMet, despite the lack of overall sequence conservation.
Assuntos
DNA/genética , Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/ultraestrutura , Proteínas Repressoras/ultraestrutura , DNA Metiltransferases Sítio Específica (Adenina-Específica)/ultraestrutura , Sequência de Aminoácidos/genética , Sequência Conservada/genética , DNA/ultraestrutura , Metilação de DNA/genética , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/ultraestrutura , Humanos , Ligação de Hidrogênio , Metiltransferases/ultraestrutura , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Conformação Proteica , Conformação Proteica em Folha beta/genética , Dobramento de Proteína , Proteína-Arginina N-Metiltransferases/genética , RNA/genética , RNA/ultraestrutura , Proteínas Repressoras/genética , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genéticaRESUMO
The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 Å structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the γ subunit complexed to α3ß3 of F1-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5'-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.
Assuntos
Domínio AAA , Enzimas de Restrição do DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Microscopia Crioeletrônica , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/isolamento & purificação , Enzimas de Restrição do DNA/ultraestrutura , DNA Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Guanosina Trifosfato/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Relação Estrutura-AtividadeRESUMO
Comprehensive understanding of structure and recognition properties of regulatory nucleic acid elements in real time and atomic level is highly important to devise efficient therapeutic strategies. Here, we report the establishment of an innovative biophysical platform using a dual-app nucleoside analog, which serves as a common probe to detect and correlate different GQ structures and ligand binding under equilibrium conditions and in 3D by fluorescence and X-ray crystallography techniques. The probe (SedU) is composed of a microenvironment-sensitive fluorophore and an excellent anomalous X-ray scatterer (Se), which is assembled by attaching a selenophene ring at 5-position of 2'-deoxyuridine. SedU incorporated into the loop region of human telomeric DNA repeat fluorescently distinguished subtle differences in GQ topologies and enabled quantify ligand binding to different topologies. Importantly, anomalous X-ray dispersion signal from Se could be used to determine the structure of GQs. As the probe is minimally perturbing, a direct comparison of fluorescence data and crystal structures provided structural insights on how the probe senses different GQ conformations without affecting the native fold. Taken together, our dual-app probe represents a new class of tool that opens up new experimental strategies to concurrently investigate nucleic acid structure and recognition in real time and 3D.
Assuntos
Desoxiuridina/análogos & derivados , Corantes Fluorescentes/química , Quadruplex G , Sondas de Ácido Nucleico/química , Compostos Organosselênicos/química , Cristalografia por Raios X , Desoxiuridina/química , Humanos , Ligantes , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Telômero/químicaRESUMO
McrBC is one of the three modification-dependent restriction enzymes encoded by the Escherichia coli K12 chromosome. Amongst restriction enzymes, McrBC and its close homologues are unique in employing the AAA+ domain for GTP hydrolysis-dependent activation of DNA cleavage. The GTPase activity of McrB is stimulated by the endonuclease subunit McrC. It had been reported previously that McrB and McrC subunits oligomerise together into a high molecular weight species. Here we conclusively demonstrate using size exclusion chromatography coupled multi-angle light scattering (SEC-MALS) and images obtained by electron cryomicroscopy that McrB exists as a hexamer in solution. Furthermore, based on SEC-MALS and SAXS analyses of McrBC and the structure of McrB, we propose that McrBC is a complex of two McrB hexamers bridged by two subunits of McrC, and that the complete assembly of this complex is integral to its enzymatic activity. We show that the nucleotide-dependent oligomerisation of McrB precedes GTP hydrolysis. Mutational studies show that, unlike other AAA+ proteins, the catalytic Walker B aspartate is required for oligomerisation.
Assuntos
Enzimas de Restrição do DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Microscopia Crioeletrônica , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Guanosina Trifosfato/metabolismo , Mutação , Multimerização Proteica , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Endonucleolytic cleavage of DNA by Type III restriction-modification (RM) enzymes requires long-range communication between at least two recognition sites in inverted orientation. This results in convergence of two nuclease domains, one each from the enzymes loaded at the recognition sites with one still bound to the site. The nucleases catalyze scission of the single-strands leading to double-strand DNA break. An obscure feature of the Type III RM enzymes EcoP1I and EcoP15I is their ability to cleave DNA having a single recognition site under certain conditions. Here we demonstrate that single-site cleavage is the result of cooperation between an enzyme bound to the recognition site in cis and one in trans. DNA cleavage is catalyzed by converging nucleases that are activated by hydrolysis-competent ATPase in presence of their respective DNA substrates. Furthermore, a single activated nuclease cannot nick a strand on its own, and requires the partner. Based on the commonalities in the features of single-site and two-site cleavage derived from this study, we propose that their mechanism is similar. Furthermore, the products of two-site cleavage can act as substrates and activators of single-site cleavage. The difference in the two modes lies in how the two cooperating enzymes converge, which in case of single-site cleavage appears to be via 3D diffusion.
Assuntos
Adenosina Trifosfatases/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/química , DNA/metabolismo , Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo III/genética , MutaçãoRESUMO
Engineering restriction enzymes with new sequence specificity has been an unaccomplished challenge, presumably because of the complexity of target recognition. Here we report detailed analyses of target recognition by Type ISP restriction-modification enzymes. We determined the structure of the Type ISP enzyme LlaGI bound to its target and compared it with the previously reported structure of a close homologue that binds to a distinct target, LlaBIII. The comparison revealed that, although the two enzymes use almost a similar set of structural elements for target recognition, the residues that read the bases vary. Change in specificity resulted not only from appropriate substitution of amino acids that contacted the bases but also from new contacts made by positionally distinct residues directly or through a water bridge. Sequence analyses of 552 Type ISP enzymes showed that the structural elements involved in target recognition of LlaGI and LlaBIII were structurally well-conserved but sequentially less-conserved. In addition, the residue positions within these structural elements were under strong evolutionary constraint, highlighting the functional importance of these regions. The comparative study helped decipher a partial consensus code for target recognition by Type ISP enzymes.
Assuntos
Proteínas de Bactérias/química , Enzimas de Restrição do DNA/química , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , DNA/química , Ligação de Hidrogênio , Lactococcus lactis/enzimologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Especificidade por SubstratoRESUMO
The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of â¼30 bp.
Assuntos
Clivagem do DNA , Enzimas de Restrição do DNA/metabolismo , DNA/química , DNA/metabolismo , Pegada de DNA , Enzimas de Restrição do DNA/química , Ativação Enzimática , Exodesoxirribonucleases , Modelos MolecularesRESUMO
Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission.
Assuntos
Proteínas de Bactérias/química , DNA Helicases/química , DNA/química , Endonucleases/química , Lactococcus lactis/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Clivagem do DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Lactococcus lactis/enzimologia , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Chi and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease. Here, we report the crystal structure of AddAB bound to DNA. The structure allows identification of a putative Chi-recognition site in an inactivated helicase domain of the AddB subunit. By generating mutant protein complexes that do not respond to Chi, we show that residues responsible for Chi recognition are located in positions equivalent to the signature motifs of a conventional helicase. Comparison with the related RecBCD complex, which recognizes a different Chi sequence, provides further insight into the structural basis for sequence-specific ssDNA recognition. The structure suggests a simple mechanism for DNA break processing, explains how AddAB and RecBCD can accomplish the same overall reaction with different sets of functional modules and reveals details of the role of an Fe-S cluster in protein stability and DNA binding.
Assuntos
DNA Helicases/química , Desoxirribonucleases/química , Exodesoxirribonucleases/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Exodesoxirribonuclease V/química , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Superfamily 1B (SF1B) helicases translocate in a 5'-3' direction and are required for a range of cellular activities across all domains of life. However, structural analyses to date have focused on how SF1A helicases achieve 3'-5' movement along nucleic acids. We present crystal structures of the complex between the SF1B helicase RecD2 from Deinococcus radiodurans and ssDNA in the presence and absence of an ATP analog. These snapshots of the reaction pathway reveal a nucleotide binding-induced conformational change of the two motor domains that is broadly reminiscent of changes observed in other SF1 and SF2 helicases. Together with biochemical data, the structures point to a step size for translocation of one base per ATP hydrolyzed. Moreover, the structures also reveal a mechanism for nucleic acid translocation in the 5'-3' direction by SF1B helicases that is surprisingly different from that of 3'-5' translocation by SF1A enzymes, and explains the molecular basis of directionality.
Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Deinococcus/enzimologia , Trifosfato de Adenosina/análogos & derivados , Cristalografia por Raios X , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
The molecular mechanism of superfamily 1Balpha helicases remains unclear. We present here the crystal structure of the RecD2 helicase from Deinococcus radiodurans at 2.2-A resolution. The structure reveals the folds of the 1B and 2B domains of RecD that were poorly ordered in the structure of the Escherichia coli RecBCD enzyme complex reported previously. The 2B domain adopts an SH3 fold which, although common in eukaryotes, is extremely rare in bacterial systems. In addition, the D. radiodurans RecD2 structure has aided us in deciphering lower resolution (3.6 A) electron density maps for the E. coli RecBCD enzyme in complex with a long DNA substrate that interacts with the RecD subunit. Taken together, these structures indicated an important role for the 1B domain of RecD, a beta-hairpin that extends from the surface of the 1A domain and interacts with the DNA substrate. On the basis of these structural data, we designed a mutant RecD2 helicase that lacks this pin. The 'pin-less' mutant protein is a fully active ssDNA-dependent ATPase but totally lacks helicase activity.
Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , DNA/metabolismo , Deinococcus/enzimologia , Exodesoxirribonuclease V/química , Exodesoxirribonuclease V/metabolismo , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
In Escherichia coli, RecBCD processes double-stranded DNA breaks during the initial stages of homologous recombination. RecBCD contains helicase and nuclease activities, and unwinds and digests the blunt-ended DNA until a specific eight-nucleotide sequence, Chi, is encountered. Chi modulates the nuclease activity of RecBCD and results in a resected DNA end, which is a substrate for RecA during subsequent steps in recombination. RecBCD also acts as a defence mechanism against bacteriophage infection by digesting linear viral DNA present during virus replication or resulting from the action of restriction endonucleases. To avoid this fate, bacteriophage lambda encodes the gene Gam whose product is an inhibitor of RecBCD. Gam has been shown to bind to RecBCD and inhibit its helicase and nuclease activities. We show that Gam inhibits RecBCD by preventing it from binding DNA. We have solved the crystal structure of Gam from two different crystal forms. Using the published crystal structure of RecBCD in complex with DNA we suggest models for the molecular mechanism of Gam-mediated inhibition of RecBCD. We also propose that Gam could be a mimetic of single-stranded, and perhaps also double-stranded, DNA.