Mechanism Of DNA Cleavage Research Articles

The fragment structure of a putative HsdR subunit of a type I restriction enzyme from Vibrio vulnificus YJ016: implications for DNA restriction and translocation activity

Among four types of bacterial restriction enzymes that cleave a foreign DNA depending on its methylation status, type I enzymes composed of three subunits are interesting because of their unique DNA cleavage and translocation mechanisms performed by the restriction subunit (HsdR). The elucidated N-terminal fragment structure of a putative HsdR subunit from Vibrio vulnificus YJ016 reveals three globular domains. The nucleolytic core within an N-terminal nuclease domain (NTD) is composed of one basic and three acidic residues, which include a metal-binding site. An ATP hydrolase (ATPase) site at the interface of two RecA-like domains (RDs) is located close to the probable DNA-binding site for translocation, which is far from the NTD nucleolytic core. Comparison of relative domain arrangements with other functionally related ATP and/or DNA complex structures suggests a possible translocation and restriction mechanism of the HsdR subunit. Furthermore, careful analysis of its sequence and structure implies that a linker helix connecting two RDs and an extended region within the nuclease domain may play a central role in switching the DNA translocation into the restriction activity.

DNA cleavage promoted by diethylenetriaminepentaacetic acid (DTPA) derivatives

DTPA and its two derivatives were synthesized and used as catalysts in DNA cleavage. Under physiological conditions, a series of experiments have been done. The effects of DNA cleavage with three ligands were studied under different concentrations, cleavage time, and pH values. The results strongly suggested that the plasmid DNA (pUC 19) can be cleaved efficiently by CITC-DTPA. For the cleavage reaction catalyzed by CITC-DTPA, Form I DNA could convert to Form II mostly, and the DNA-cleavage mechanism involved an oxidative pathway.

Bisintercalator-containing dinuclear iron(III) complex: An efficient artificial nuclease

Two acridine groups were successfully introduced into di-iron(III) complex. DNA cleavage experiments indicated that complex conjugating bisacridine groups can enhance 300-fold for the cleavage efficiency compared with complex lacking of acridine conjugation. Further ligation assay of DNA segments provided the evidence for hydrolytic mechanism of DNA cleavage.

Copper(II) complexes of methylated glycine derivatives: Effect of methyl substituent on their DNA binding and nucleolytic property

A set of copper(II) complexes of glycine and methylated glycine derivatives, Cu(aa) 2, consisting of C-dimethylglycine, l-alanine, N-dimethylglycine and sarcosine, was investigated for their DNA binding and nucleolytic properties by means of EPR and visible spectroscopy, and electrophoresis. They bind weakly to DNA with apparent binding constants in the range 1.8–2.9 × 10 3 M −1 with very similar orientation. No DNA cleavage is observed in the absence of exogenous agents. Copper(II) complexes of N-methylated derivatives bind to DNA more stereo-specifically and less strongly, and their oxidative DNA cleavage is less efficient than those of the corresponding C-methylated derivatives in the presence of hydrogen peroxide (H 2O 2) alone, or sodium ascorbate (NaHA) alone or tandem H 2O 2–NaHA. The oxidative DNA cleavage mechanism in the three systems involves a common copper(I) species. Neocuproine can inhibit DNA cleavage by these complexes.

Ruthenium(II) Complexes of Bipyridine−Glycoluril and their Interactions with DNA

Nine complexes of the type [Ru(N-N)(2)(BPG)]Cl(2) 1-4, [Ru(N-N)(BPG)(2)]Cl(2) 5-8, and [Ru(BPG)(3)]Cl(2) 9 where N-N is 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz), which incorporates bipyridine-glycoluril (BPG-4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f][1,10]phenanthroline-6,13-dione) as the ancillary ligand, have been synthesized and characterized. These complexes with the peripheral polypyridyl ligands have the ability to form conjugates with DNA. The DNA binding (absorption spectroscopy, steady-state and time-resolved emission measurements, steady-state emission quenching measurements) and cleavage (under dark and irradiated conditions) by these complexes has been studied to investigate the influence of the ancillary ligand. The binding ability of these complexes to DNA is dependent on the planarity of the intercalative polypyridyl ligand, which is further affected by the ancillary bipyridine-glycoluril ligand. The complexes 3, 4, 7, and 8 bind to CT-DNA with binding constants on the order of 10(4) M(-1). Time-resolved emission measurements on the DNA-bound complexes 1, 3, 5-7, and 9 show monoexponential decay of the excited states, whereas complexes 2, 4, and 8 show biexponential decay with short- and long-lived components. Interaction of complexes 2-9 with plasmid pBR322 DNA studied by gel electrophoresis experiments reveals that all complexes cleave DNA efficiently at micromolar concentrations under dark and anaerobic conditions probably by a hydrolytic mechanism. Complexes 3, 4, 7, 8, and [Ru(bpy)(2)(dppz)](2+) show extensive DNA cleavage in the presence of light with a shift in mobility of form I of DNA probably due to the high molecular weight of DNA-complex conjugates. However, the extent of the cleavage is augmented on irradiation in the case of complexes 3, 4, 7, and 8, which include the planar dpq and dppz ligands, suggesting a combination of hydrolytic and oxidative mechanism for the DNA scission. Molecular mechanics calculations of these systems corroborate the DNA binding and cleavage mechanisms.

Enhancement of DNA cleavage activity of an unnatural ferrocene-amino acid conjugate

The nucleolytic activity of an unnatural ferrocenyl amino acid can be significantly enhanced under reducing or Fenton conditions while exploiting different mechanisms of DNA cleavage. In addition, results from high resolution electrophoresis indicate that the ferrocenyl amino acid exhibits non-sequence specific cleavage of the DNA backbone.

Topo2008: DNA Topoisomerases in Biology and Medicine

Topo2008: DNA Topoisomerases in Biology and Medicine

Synthesis, X-ray crystal structures, magnetism, and DNA cleavage properties of copper(ii) complexes with 1,4-tpbd ligand

Four new copper(II) complexes, [Cu(1,4-tpbd)Br(2)] (), [Cu(2)(1,4-tpbd)(H(2)O)(4)](ClO(4))(4) (), [Cu(2)(1,4-tpbd)(1,10-phen)(2)(DMF)(2)](ClO(4))(4) () and [Cu(2)(1,4-tpbd)(2,2'-bpy)(2)(ClO(4))(2)](ClO(4))(2) (), [1,4-tpbd = N,N,N',N'-tetrakis(2-pyridylmethyl)benzene-1,4-diamine], have been synthesized to serve as artificial nucleases. Single crystal X-ray diffraction reveals that the copper(II) atom has a distorted intermediate between square pyramidal and trigonal bipyramidal configuration for and a distorted square pyramidal geometry for , while a distorted octahedral environment for and . Variable-temperature magnetic susceptibility studies (2-300 K) indicate the existence of antiferromagnetic coupling between the copper(II) ions in complex . The interactions of the four complexes with calf thymus DNA (CT-DNA) have been investigated by UV absorption, fluorescent spectroscopy and cyclic voltammetry, and the modes of CT-DNA binding for the complexes have been proposed. In the absence of external agents, supercoiled plasmid DNA cleavage by the complexes was performed under aerobic conditions, the influence on the DNA cleavage process of different complex concentrations, reaction times was also studied. The DNA cleavage mechanisms were demonstrated with radical scavengers and anaerobic conditions, indicating all complexes cleaved pBR322 DNA in 50 mM Tris-HCl/18 mM NaCl buffer (pH = 7.2) at 37 degrees C through a hydrolytic process. In the four copper(II) complexes, complex showed highest cleavage activity with the pseudo-Michaelis-Menten kinetic parameters k(cat) = 4.23 h(-1) and K(m) = 2.4 x 10(-5) M.

Molecular Dynamics Simulations on Binding Models of Dervan-Type Polyamide + Cu(II) Nuclease Ligands to DNA

Molecular dynamics simulations for the ligand [Cu(BPA)](2+) (BPA = bis(2-pyridylmethyl)amine) nuclease bound to either the single-chain polyamide, ImPyImImPybetaDp, or the antiparallel double-polyamide, (ImImImbetaDp)(2), and associated with DNA were performed to predict the improvement of selective DNA cleavage ability of copper-based chemical nucleases. The results from the simulations indicate that either polyamide-bound [Cu(BPA)](2+) + OOH(-) (which is a necessary substrate in the redox mechanism of DNA cleavage) can locate in the minor groove of DNA in the parallel orientation similar to the X-ray structure. As a consequence of the polyamide + [Cu(BPA)](2+) + OOH(-) ligand binding to DNA, the active end oxygen atom of the OOH(-) substrate is held in close proximity to the known target C1'H or C4'H protons of the DNA. Upon examinations of six different ligands binding to DNA, the binding interaction of the entire ligand with DNA for each polyamide increases with the presence of [Cu(BPA)](2+) + OOH(-) ligand. The N-H groups of the linking regions from polyamide play an important role for the interaction by functioning as H-bond donors to N or O atoms of the nucleobase located on the floor of the minor groove of DNA. The investigation provides the feasible protocol to improve the selective DNA cleavage activity of chemical nucleases assisted by DNA recognition agents.

Molecular mechanism for generation of antibody memory

Activation-induced cytidine deaminase (AID) is the essential enzyme inducing the DNA cleavage required for both somatic hypermutation and class switch recombination (CSR) of the immunoglobulin gene. We originally proposed the RNA-editing model for the mechanism of DNA cleavage by AID. We obtained evidence that fulfils three requirements for CSR by this model, namely (i) AID shuttling between nucleus and cytoplasm, (ii) de novo protein synthesis for CSR, and (iii) AID-RNA complex formation. The alternative hypothesis, designated as the DNA-deamination model, assumes that the in vitro DNA deamination activity of AID is representative of its physiological function in vivo. Furthermore, the resulting dU was removed by uracil DNA glycosylase (UNG) to generate a basic site, followed by phosphodiester bond cleavage by AP endonuclease. We critically examined each of these provisional steps. We identified a cluster of mutants (H48A, L49A, R50A and N51A) that had particularly higher CSR activities than expected from their DNA deamination activities. The most striking was the N51A mutant that had no ability to deaminate DNA in vitro but retained approximately 50 per cent of the wild-type level of CSR activity. We also provide further evidence that UNG plays a non-canonical role in CSR, namely in the repair step of the DNA breaks. Taking these results together, we favour the RNA-editing model for the function of AID in CSR.

Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.

Dissociation of in vitro DNA deamination activity and physiological functions of AID mutants

Activation-induced cytidine deaminase (AID) is essential for the DNA cleavage that initiates both somatic hypermutation (SHM) and class switch recombination (CSR) of the Ig gene. Two alternative mechanisms of DNA cleavage by AID have been proposed: RNA editing and DNA deamination. In support of the latter, AID has DNA deamination activity in cell-free systems that is assumed to represent its physiological function. To test this hypothesis, we generated various mouse AID mutants and compared their DNA deamination, CSR, and SHM activities. Here, we compared DNA deamination, CSR, and SHM activities of various AID mutants and found that most of their CSR or SHM activities were disproportionate with their DNA deamination activities. Specifically, we identified a cluster of mutants (H48A, L49A, R50A, and N51A) with low DNA deamination activity but relatively intact CSR activity. Of note is an AID mutant (N51A) that retained CSR function but lost DNA deamination activity. In addition, an APOBEC1 mutation at N57, homologous to N51 of AID, also abolished DNA deamination activity but retained RNA editing activity. These results indicate that DNA deamination activity does not represent the physiological function of AID.

Insights into the DNA cleavage mechanism of human LINE‐1 retrotransposon endonuclease

The human LINE-1 endonuclease (L1-EN) contributes in defining the genomic integration sites of the abundant human L1 and Alu retrotransposons. LINEs have been considered as possible vehicles for gene delivery and understanding the mechanism of L1-EN could help engineering them as genetic tools. We tested the in vitro activity of point mutants in three L1-EN residues--Asp145, Arg155, Ile204--that are key for DNA cleavage, and determined their crystal structures. The L1-EN structure remains overall unaffected by the mutations, which change the enzyme activity but leave DNA cleavage sequence specificity mostly unaffected. To better understand the mechanism of L1-EN, we performed molecular dynamics simulations using as model the structures of wild type EN-L1, of two betaB6-betaB5 loop exchange mutants we have described previously to be important for DNA recognition, of the R155A mutant from this study, and of the homologous TRAS1 endonuclease: all confirm a rigid scaffold. The simulations crucially indicate that the betaB6-betaB5 loop shows an anticorrelated motion with the surface loops betaA6-betaA5 and betaB3-alphaB1. The latter loop harbors N118, a residue that alters DNA cleavage specificity in homologous endonucleases, and implies that the plasticity and correlated motion of these loops has a functional importance in DNA recognition and binding. To further explore how these loops are possibly involved in DNA binding, we docked computationally two DNA substrates to our structure, one involving a flipped-out nucleotide downstream the scissile phosphodiester; and one not. The models for both scenarios are feasible and agree with the hypotheses derived from the dynamic simulations. The reduced cleavage activity we have observed for the I204Y mutant above however, favors the flipped out nucleotide model.

On the distinction of the mechanisms of DNA cleavage by restriction enzymes—The I-, II-, and III-type molecular motors

A comparative physical description is given for the functioning of various restriction enzymes and for their processes of DNA cleavage. The previously proposed model system of kinetic equations is applied to the I-and III-type enzymes, which use ATP molecules as an energy source, while the II-type enzymes work thanks to catalytic reactions with participation of an electric field. All the enzymes achieved bending and twisting DNA, providing for either the linear motion of the II-type enzyme along the DNA chain or the DNA translocation by the I-and III-type enzymes due to moving chiral kinks. A comparative estimation of the considered linear and angular velocities is performed. The role of stalling forces for enzyme-DNA complexes, which induce the observed cutting of the DNA either inside the enzyme (II) or in some “weak” places outside enzymes I and III, which results in the supercoiling of the DNA, is shown. The role of ionic screening for the described processes is discussed.

DNA Binding and Cleaving Activity of the New Cleft Molecule N,N′-Bis(guanidinoethyl)-2,6-pyridinedicarboxamide in the Absence or in the Presence of Copper(II)

Novel cleft molecule pyridine-2,6-dicarboxamide appending two guanidinoethyl group side arms (Gua) was synthesized. The interactions of the cleft molecule in the absence of copper(II) (Gua) or in the presence of copper(II) (Cu2+-Gua) with calf thymus DNA were studied by fluorescence and CD spectroscopy. The results indicate that the DNA binding affinity of Cu2+-Gua is stronger than that of Gua, and the binding constants of Cu2+-Gua and Gua are 1.61 x 10(6) M(-1) and 2.86 x 10(5) M(-1), respectively. Agarose gel electrophoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of Gua and Cu2+-Gua. Kinetic data of DNA cleavage promoted by Cu2+-Gua under physiological conditions fit a saturation kinetic profile with k(max) of 0.0173 +/- 0.0011 h(-1), which gave a aproximately 10(6)-fold rate acceleration over uncatalyzed supercoiled DNA, while the catalyst concentration is lower than 0.0625 mM. The hydrolysis pathway was proposed as the possible mechanism for DNA cleavage promoted by Cu2+-Gua. The acceleration is due to efficient cooperative catalysis of the copper cation center and the functional groups (bis(guanidinium) groups).

Human topoisomerase II uses a two-metal-ion mechanism for DNA cleavage

The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen.

Putative Functional Domains of Human Cytomegalovirus pUL56 Involved in Dimerization and Benzimidazole D-Ribonucleoside Activity

Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89 (the large and small subunits of the HCMV terminase, respectively) their mechanism of action is not yet fully understood. We aimed here to better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives. The HCMV pUL56 protein was studied by sequence analysis of the HCMV UL56 gene and herpesvirus counterparts combined with primary structure analysis of the corresponding amino acid sequences. The UL56 sequence analysis of 45 HCMV strains and counterparts among herpesviruses allowed the identification of 12 conserved regions. Moreover, comparison with the product of gene 49 (gp49) of bacteriophage T4 suggested that the pUL56 zinc finger is localized close to the dimerization site of pUL56, providing a spatial organization of the catalytic site that allows recognition and cleavage of DNA. This study provides a basis to investigate the mechanism of concatemeric DNA cleavage and a biochemical basis for DNA packaging inhibition by benzimidazole derivatives.

Ferrocene‐bridging dinuclear cyclen copper(II) complexes as high efficient artificial nucleases: design, synthesis and interaction with DNA

AbstractTwo novel cyclen copper(II) complexes bridged by ferrocene were designed and synthesized. Both of these complexes exhibited excellent cleavage ability towards plasmid DNA via an oxidative pathway without the presence of any additives. Cyclic voltammetry was used to investigate the electrochemistry characters of the interaction between the complexes and DNA. Agarose gel electrophoresis was carried out to study the DNA restriction ability of these complexes, and the results indicated that the complexes showed higher cleavage efficiency via an oxidative pathway without the presence of any additives. The mechanism of DNA cleavage catalyzed by these complexes was examined by the addition of various scavengers, and the results showed that singlet oxygen and hydroxyl radical might be responsible for the cleavage process. Copyright © 2008 John Wiley & Sons, Ltd.

DNA cleavage promoted by 2,9-dimethyl-4,7-diazadecane-2,9-dithiol (DDD) derivatives

Three piperidine derivatives of 2,9-dimethyl-4,7-diazadecane-2,9-dithiol (DDD), NEPDDD, NEMPDDD, and NEMMPDDD, were synthesized and used as catalysts in DNA cleavage. Under physiological conditions, a series of experiments have been done. The effects of DNA cleavage with three ligands were studied under different concentrations, cleavage time, and pH values. The results strongly suggested that the plasmid DNA (pUC 19) can be cleaved efficiently by these ligands. For the cleavage reaction catalyzed by NEMPDDD, Form I DNA could convert to Form II completely, and the DNA-cleavage mechanism involved an oxidative pathway.

The first dinuclear copper(ii) and zinc(ii) complexes containing novel Bis-TACN: syntheses, structures, and DNA cleavage activities

Two novel binuclear complexes [Cu(2)(L)].(ClO(4))(2) (1) and [Zn(2)(L)].(ClO(4))(2) (2) were synthesized and crystallographically characterized {L = 1(4),5(4)-dimethyl-1(2),5(2)-dihydroxy-1(1,3),5(1,3)-dibenzene-3(1,4),7(1,4)-di-1,4,7-triazacyclononane}. The cation [Cu(2)(L)](2+) structure of 1 is similar to that of [Zn(2)(L)](2+) of 2. The central ion is bridged by the di-phenoxo of L and lies in a close to perfect square pyramidal geometry. 1 and 2 crystallize in the triclinic space group P1. The two complexes effectively promote the cleavage of plasmid DNA in the presence of activating agents at physiological pH and temperature. The pseudo-Michaelis-Menten kinetic parameters k(cat) = 1.61 h(-1), K(m) = 1.35 x 10(-5) M for complex 1 in the presence of mercaptoethanol; k(cat) = 2.48 h(-1), K(m) = 5.5 x 10(-5)M for complex 2 in the presence of hydrogen peroxide were obtained. The mechanism of plasmid DNA cleavage was studied by adding standard radical scavengers. DNA cleavage reaction by the binuclear Zn(II)/H(2)O(2) system is a hydrolytic mechanism.