Further purification and characterization of T4 endonuclease V.

T4 endonuclease V, which is involved in repair of ultraviolet-damaged DNA, has been purified 3600 fold from T4D-infected Escherichia coli. The enzyme shows optimal activity at pH 7.2 and does not require added divalent ions. Endonuclease V attacks both native and heat-denatured DNA provided that the DNA has been irradiated, and the enzyme activity is dependent on the dose of ultraviolet irradiation. The rate and the extent of the reaction are greater with irradiated native DNA although the Km values for the two types of DNA are the same (2.25 - 10(-5) M). The enzyme is readily inactivated by heat and is sensitive to p-chloromercuribenzoate. Endonuclease V-treated irradiated DNA is degraded by spleen phosphodiesterase only when the DNA has been treated with alkaline phosphatase, suggesting that the enzyme produces 5'-phosphoryl termini.     

A Theoretical Study of the Aqueous Hydration of Canonical B d(CGCGAATTCGCG): Monte Carlo Simulation and Comparison with Crystallographic Ordered Water Sites

Abstract

Monte Carlo computer simulation is described for the dodecamer d(CGCGAATTCGCG) together with 1777 water molecules at an environmental density of 1 gm/cc in a cubic cell under periodic boundary conditions. Water-water interactions were treated using the TIP4P potential and the solute water interactions by TIP4P spliced with the non-bonded interactions from the AMBER 3.0 force field. The simulation was subjected to proximity analysis to obtain solute coordination numbers and pair interaction energies for each solute atom. Hydration density distributions partitioned into contributions from the major groove side, the minor groove side and the sugar-phosphate backbone were examined, and the probabilities of occurence for one- and two-water bridges in the simulation were enumerated. The results were compared with observations of crystallographic ordered water sites from x-ray diffraction studies on the native dodecamer by Dickerson and coworkers.     

On the recognition and cleavage mechanism of Escherichia coli endodeoxyribonuclease V, a possible DNA repair enzyme

Escherichia coli endodeoxyribonuclease V acts at many sites of damage in duplex DNA, including apurinic/apyrimidinic sites, lesions induced by ultraviolet light which are not pyrimidine dimers, adducts of 7-bromomethylbenz[a]anthracene, and, as demonstrated earlier (Gates, F. T., and Linn, S. (1977a) J. Biol. Chem. 252. 1647-1653), it degrades uracil-containing duplex DNA most efficiently. The cleavage rate increases with increasing substitution of uracil for thymine in T5 DNA, with a replacement of one-eight of thymine generating the apparent maximum cleavage rate. However, the apparent reaction limit with DNA containing 3.8% of thymine replaced by uracil corresponds to cleavage at only 6% of the dUMP residues. Evidently, the enzyme recognizes some peculiarities of abnormal DNA structure, but not simply distortions, since some lesions, including pyrimidine dimers, are not substrates. Endonuclease V generates double strand breaks in a constant ratio to single strand nicks, regardless of the substrate. It degrades DNA processively, completing the digestion of one substrate molecule before proceeding to the next. The enzyme also appears to act cooperatively. Cleavage at methylbenz[a]anthracene adducts is usually or always 5' to the lesion. Endonuclease V seems well suited to act as a DNA repair enzyme, surveying the genome for structural distortions generated by lesions for which specific repair systems might not exist.     

Role of active site residues in the glycosylase step of T4 endonuclease V. Computer simulation studies on ionization states.

T4 Endonuclease V (EndoV) is a base excision repair enzyme that removes thymine dimers (TD) from damaged DNA. To elucidate the role of the active site residues in catalysis, their pK(a)'s were evaluated using the semimicroscopic version of the protein dipoles-Langevin dipoles method (PDLD/S). Contributions of different effects to the pK(a) such as charge-charge interactions, conformational rearrangement, protein relaxation, and DNA binding were analyzed in detail. Charging of the active site residues was found to be less favorable in the complex than in the free enzyme. The pK(a) of the N-terminus decreased from 8.01 in the free enzyme to 6.52 in the complex, while the pK(a) of Glu-23 increased from 1. 52 to 7.82, which indicates that the key residues are neutral in the reactant state of the glycosylase step. These pK(a)'s are in agreement with the optimal pH range of the reaction and support the N-terminus acting as a nucleophile. The Glu-23 in its protonated form is hydrogen bonded to O4' of the sugar of 5' TD and can play a role in increasing the positive charge of C1' and, hence, accelerating the nucleophilic substitution. Furthermore, the neutral Glu-23 is a likely candidate to protonate O4' to induce ring opening required to complete the glycosylase step of EndoV. The positively charged Arg-22 and Arg-26 provide an electrostatically favorable environment for the leaving base. To distinguish between S(N)1 and S(N)2 mechanisms of the glycosylase step the energetics of protonating O2 of 5' TD was calculated. The enzyme was found to stabilize the neutral thymine by approximately 3.6 kcal/mol, whereas it destabilizes the protonated thymine by approximately 6.6 kcal/mol with respect to an aqueous environment. Consequently, the formation of a protonated thymine intermediate is unlikely, indicating an S(N)2 reaction mechanism for the glycosylase step.     

Structural Insights Into DNA Repair by RNase T—An Exonuclease Processing 3′ End of Structured DNA in Repair Pathways

DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3′ end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3′ end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3′ overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V–dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3′-end processing.     

Structure and hydration of BamHI DNA recognition site: a molecular dynamics investigation

The results of a 3-ns molecular dynamics simulation of the dodecamer duplex d(TATGGATCCATA)(2) recognized by the BamHI endonuclease are presented here. The DNA has been simulated as a flexible molecule using an AMBER force field and the Ewald summation method, which eliminates the undesired effects of truncation and permits evaluation of the full effects of electrostatic forces. The starting B conformation evolves toward a configuration quite close to that observed through x-ray diffraction in its complex with BamHI. This configuration is fairly stable and the Watson-Crick hydrogen bonds are well maintained over the simulation trajectory. Hydration analysis indicates a preferential hydration for the phosphate rather than for the ester oxygens. Hydration shells in both the major and minor groove were observed. In both grooves the C-G pairs were found to be more hydrated than A-T pairs. The "spine of hydration" in the minor groove was clear. Water residence times are longer in the minor groove than in the major groove, although relatively short in both cases. No special long values are observed for sites where water molecules were observed by x-ray diffraction, indicating that water molecules having a high probability of being located in a specific site are also fast-exchanging.     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light

Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme--DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. The assay is simple, rapid, and specific, which makes it useful for detecting T4 endonuclease V activity both in crude lysates and in purified preparations. We have used it to monitor enzyme activity during purification and to study binding of the enzyme to DNA under conditions that minimize the ability of the enzyme to nick DNA. From our data we conclude that (1) T4 endonuclease V binds to UV-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme--DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme--DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline--citrate.     

Repair of deaminated bases in DNA

Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil, hypoxanthine, and xanthine. When cells are under oxidative stress that is induced either by oxidizing agents or by mitochondrial dysfunction, additional deamination products such as 5-hydroxymethyluracil (5-HMU) and 5-hydroxyuracil (5-OH-Ura) are formed. The cellular level of these highly mutagenic lesions is increased substantially when cells are exposed to DNA damaging agent, such as ionizing radiation, redox reagents, nitric oxide, and others. The cellular repair of deamination products is predominantly through the base excision repair (BER) pathway, a major cellular repair pathway that is initiated by lesion specific DNA glycosylases. In BER, the lesions are removed by the combined action of a DNA glycosylase and an AP endonuclease, leaving behind a one-base gap. The gapped product is then further repaired by the sequential action of DNA polymerase and DNA ligase. DNA glycosylases that recognize uracil, 5-OH-Ura, 5-HMU (derived from 5-methylcytosine) and a T/G mismatch (derived from a 5-methylcytosine/G pair) are present in most cells. Many of these glycosylases have been cloned and well characterized. In yeast and mammalian cells, hypoxanthine is efficiently removed by methylpurine N-glycosylase, and it is thought that BER might be an important pathway for the repair of hypoxanthine. In contrast, no glycosylase that can recognize xanthine has been identified in either yeast or mammalian cells. In Escherichia coli, the major enzyme activity that initiates the repair of hypoxanthine and xanthine is endonuclease V. Endonuclease V is an endonuclease that hydrolyzes the second phosphodiester bond 3' to the lesion. It is hypothesized that the cleaved DNA is further repaired through an alternative excision repair (AER) pathway that requires the participation of either a 5' endonuclease or a 3'-5' exonuclease to remove the damaged base. The repair process is then completed by the sequential actions of DNA polymerase and DNA ligase. Endonuclease V sequence homologs are present in all kingdoms, and it is conceivable that endonuclease V might also be a major enzyme that initiates the repair of hypoxanthine and xanthine in mammalian cells.     

Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations

Endonuclease V (endo V) recognizes a broad range of aberrations in DNA such as deaminated bases or mismatches. It nicks DNA at the second phosphodiester bond 3′ to a deaminated base or a mismatch. Endonuclease V obtained from Thermotoga maritima preferentially cleaves purine mismatches in certain sequence context. Endonuclease V has been combined with a high-fidelity DNA ligase to develop an enzymatic method for mutation scanning. A biochemical screening of site-directed mutants identified mutants in motifs III and IV that altered the base preferences in mismatch cleavage. Most profoundly, a single alanine substitution at Y80 position switched the enzyme to essentially a C-specific mismatch endonuclease, which recognized and cleaved A/C, C/A, T/C, C/T and even the previously refractory C/C mismatches. Y80A can also detect the G13D mutation in K-ras oncogene, an A/C mismatch embedded in a G/C rich sequence context that was previously inaccessible using the wild-type endo V. This investigation offers insights on base recognition and active site organization. Protein engineering in endo V may translate into better tools in mutation recognition and cancer mutation scanning.     

Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis.

Endonuclease IV is the archetype for a conserved apurinic/apyrimidinic (AP) endonuclease family that primes DNA repair synthesis by cleaving the DNA backbone 5' of AP sites. The crystal structures of Endonuclease IV and its AP-DNA complex at 1.02 and 1.55 A resolution reveal how an alpha8beta8 TIM barrel fold can bind dsDNA. Enzyme loops intercalate side chains at the abasic site, compress the DNA backbone, bend the DNA approximately 90 degrees, and promote double-nucleotide flipping to sequester the extrahelical AP site in an enzyme pocket that excludes undamaged nucleotides. These structures suggest three Zn2+ ions directly participate in phosphodiester bond cleavage and prompt hypotheses that double-nucleotide flipping and sharp bending by AP endonucleases provide exquisite damage specificity while aiding subsequent base excision repair pathway progression.     

Multiple cleavage activities of endonuclease V from Thermotoga maritima: recognition and strand nicking mechanism

Endonuclease V is a deoxyinosine 3'-endonuclease which initiates removal of inosine from damaged DNA. A thermostable endonuclease V from the hyperthermophilic bacterium Thermotoga maritima has been cloned and expressed in Escherichia coli. The DNA recognition and reaction mechanisms were probed with both double-stranded and single-stranded oligonucleotide substrates which contained inosine, abasic site (AP site), uracil, or mismatches. Gel mobility shift and kinetic analyses indicate that the enzyme remains bound to the cleaved inosine product. This slow product release may be required in vivo to ensure an orderly process of repairing deaminated DNA. When the enzyme is in excess, the primary nicked products experience a second nicking event on the complementary strand, leading to a double-stranded break. Cleavage at AP sites suggests that the enzyme may use a combination of base contacts and local distortion for recognition. The weak binding to uracil sites may preclude the enzyme from playing a significant role in repair of such sites, which may be occupied by uracil-specific DNA glycosylases. Analysis of cleavage patterns of all 12 natural mismatched base pairs suggests that purine bases are preferrentially cleaved, showing a general hierarchy of A = G > T > C. A model accounting for the recognition and strand nicking mechanism of endonuclease V is presented.     

A Simple Solvation Model Along with A Multibody Dynamics Strategy (MBO(N)D) Produces Stable DNA Simulations that are Faster than Traditional Atomistic Methods

Abstract

We are developing solvation strategies that complement the speed advantage of MBO(N)D (a multibody simulation approach developed by Moldyn) for simulating biomolecular systems. In this report we propose to approximate the effect of bulk waters on DNA by using only a thin layer of waters proximate to the surface of DNA (which we will call the ‘thin shell approach’ or TSA). We will show that the TSA combined with substructuring (the grouping of atoms into rigid or flexible bodies) of the Dickerson dodecamer produces good comparisons with standard atomistic methods (over a nanosecond trajectory) as judged by a variety of DNA specific geometric (e.g., CURVES output) and dynamics (power spectra) properties. The MBO(N)D method, however, was faster than atomistic by a factor of six using the same solvation strategy and factor of 70 when compared to fully solvated atomistic system. The key to the speed of MBO(N)D is in its ability to use large time steps during dynamics. By keeping only a shell of molecules of water proximate to the dodecamer, we limit artifacts due to surface tension at the water-vacuum interface. These proximate waters are fairly immobile as compared to those in bulk and therefore do not severely limit the time step in the simulation. The strengths and limitations of this solvation approach, and future directions, will also be discussed.     

Role of base flipping in specific recognition of damaged DNA by repair enzymes

DNA repair enzymes induce base flipping in the process of damage recognition. Endonuclease V initiates the repair of cis, syn thymine dimers (TD) produced in DNA by UV radiation. The enzyme is known to flip the base opposite the damage into a non-specific binding pocket inside the protein. Uracil DNA glycosylase removes a uracil base from G.U mismatches in DNA by initially flipping it into a highly specific pocket in the enzyme. The contribution of base flipping to specific recognition has been studied by molecular dynamics simulations on the closed and open states of undamaged and damaged models of DNA. Analysis of the distributions of bending and opening angles indicates that enhanced base flipping originates in increased flexibility of the damaged DNA and the lowering of the energy difference between the closed and open states. The increased flexibility of the damaged DNA gives rise to a DNA more susceptible to distortions induced by the enzyme, which lowers the barrier for base flipping. The free energy profile of the base-flipping process was constructed using a potential of mean force representation. The barrier for TD-containing DNA is 2.5 kcal mol(-1) lower than that in the undamaged DNA, while the barrier for uracil flipping is 11.6 kcal mol(-1) lower than the barrier for flipping a cytosine base in the undamaged DNA. The final barriers for base flipping are approximately 10 kcal mol(-1), making the rate of base flipping similar to the rate of linear scanning of proteins on DNA. These results suggest that damage recognition based on lowering the barrier for base flipping can provide a general mechanism for other DNA-repair enzymes.     

T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.     

Metalloenzymes in DNA repair. Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1.

Escherichia coli endonuclease IV and its Saccharomyces cerevisiae homologue Apn1, two DNA repair enzymes for free radical damages, were previously shown to be inactivated by metal-chelating agents. In the present study, atomic absorption spectrometry of endonuclease IV revealed the presence of 2.4 zinc and 0.7 manganese atoms, whereas Apn1 contained 3.3 zinc atoms and no significant manganese. EDTA-inactivated endonuclease IV retained 0.7 zinc atom but little detectable manganese. ZnCl2 reactivated 1,10-phenanthroline-treated Apn1, but was ineffective with endonuclease IV treated with either 1,10-phenanthroline or EDTA. In contrast, enzymatic activity was restored to both enzymes after EDTA treatment by incubation with CoCl2 and to a lesser extent by MnCl2. Endonuclease IV, reactivated with CoCl2 or MnCl2, regained all of the activities characteristic of the native enzyme. MnCl2 was as effective as CoCl2 at restoring activity to the 1,10-phenanthroline-treated enzymes. The results indicate that intrinsic metals play critical roles in both endonuclease IV and Apn1 and that manganese may perform a special function in endonuclease IV. Possible mechanistic roles for the metals in these DNA repair enzymes are discussed.     

Water and ion binding around RNA and DNA (C,G) oligomers

The dynamics, hydration, and ion-binding features of two duplexes, the A(r(CG)(12)) and the B(d(CG)(12)), in a neutralizing aqueous environment with 0.25 M added KCl have been investigated by molecular dynamics (MD) simulations. The regular repeats of the same C=G base-pair motif have been exploited as a statistical alternative to long MD simulations in order to extend the sampling of the conformational space. The trajectories demonstrate the larger flexibility of DNA compared to RNA helices. This flexibility results in less well defined hydration patterns around the DNA than around the RNA backbone atoms. Yet, 22 hydration sites are clearly characterized around both nucleic acid structures. With additional results from MD simulations, the following hydration scale for C=G pairs can be deduced: A-DNA相似文献   

Dark Repair of Ultraviolet-Irradiated Deoxyribonucleic Acid by Bacteriophage T4: Purification and Characterization of a Dimer-Specific Phage-Induced Endonuclease

The purification and properties of an ultraviolet (UV) repair endonuclease are described. The enzyme is induced by infection of cells of Escherichia coli with phage T4 and is missing from extracts of cells infected with the UV-sensitive and excision-defective mutant T4V(1). The enzyme attacks UV-irradiated deoxyribonucleic acid (DNA) containing either hydroxymethylcytosine or cytosine, but does not affect native DNA. The specific substrate in UV-irradiated DNA appears to be pyrimidine dimer sites. The purified enzyme alone does not excise pyrimidine dimers from UV-irradiated DNA. However, dimer excision does occur in the presence of the purified endonuclease plus crude extract of cells infected with the mutant T4V(1).