DNA hydrolysis by inorganic catalysts

Non-enzymatic reagents that efficiently promote the hydrolytic cleavage of DNA currently receive much attention since they have many potential applications in molecular biology. This review focuses on recent progress in the hydrolysis of the phosphodiester backbone of DNA by metal ions and metal complexes. Pioneering work on the sequence-selective DNA scission by an artificial restriction enzyme, which is prepared by covalent attachment of a cerium(IV) complex to an antisense-deoxyoligonucleotide, is discussed. Received: 22 March 1999 / Received revision: 28 May 1999 / Accepted: 28 May 1999     

Double-strand DNA hydrolysis by dilanthanide complexes

 Although there has been progress in developing artificial hydrolytic DNA cleaving agents, none of these has been shown to carry out the double-strand hydrolysis of DNA. We demonstrate that La(III) or Ce(IV) combined with the ligand 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetate (HPTA) in a 2 : 1 ratio can efficiently cleave supercoiled plasmid DNA at 55  °C within a 3-h period. Analysis of end-labeled restriction fragments cleaved by these complexes reveals 3′- and 5′-ends consistent with a hydrolytic mechanism. Unlike for other polydentate carboxylate complexes, plasmid DNA cleavage by La₂(HPTA) or Ce₂(HPTA) affords a significant amount of linear DNA with a considerable fraction of the supercoiled form still remaining. This result implies that La₂(HPTA) and Ce₂(HPTA) can carry out double-strand cleavage of plasmid DNA. La₂(HPTA) and Ce₂(HPTA) represent the first metal complexes demonstrated to be capable of double-strand hydrolytic cleavage of plasmid DNA. Received: 29 March 1999 / Accepted: 9 July 1999     

Comparative studies of Feulgen hydrolysis for DNA

Summary We compared the Feulgen hydrolysis curves (37° C, 5 M HCl) of human blood lymphocytes fixed by the following four methods: 96% ethanol, 60 min at 20° C; ethanol-acetone, 11, 120 min at 4° C; ethanolglacial acetic acid mixture (31), containing 2% of formaldehyde (EAF), 90 min at 20° C; and ethanol-glacial acetic acid (31), 60 min followed by 5% chrome alum solution for 360 min at 20° C. The best results were obtained with EAF-fixation, with respect to the highest amount of DNA-Schiff complex at the peak point of the curve, the longest plateau region and the smallest scattering of DNA-Schiff amount values along the plateau. With other types of fixation the addition of polyethylene glycol (PEG) 6,000 to the hydrolysis solution resulted in modification of the shape of the hydrolysis curve so that it became nearly similar to the EAF-curve. The effect of PEG₆₀₀₀ on the EAF-curve was minimal. Slides fixed by ethanol were used for a comparison of polyethylene glycols with m.w. 1,500, 6,000 and 20,000. The longest plateau was obtained with PEG₆₀₀₀. The only effect of PEG₁₅₀₀ was a dramatic increase of DNA-Schiff amount at the peak point. PEG₂₀₀₀₀ had no significant effect on the shape of the hydrolysis curve. The results are discussed in terms of Kjellstrand's chain with a stable structure model of Feulgen hydrolysis.     

DNA hydrolysis by rare-earth metal ions.

Plasmid DNA and poly(dA) are cleaved by rare-earth(III) ions at pH 7-8 and 50 degrees C. The cleavage has been confirmed by prompt conversion of supercoiled pBR 322 plasmid DNA (Form I) to a relaxed Form II. Furthermore, degradation of poly(dA) to shorter oligonucleotides is clearly evidenced by HPLC. A possible application of the metal ions (and their complexes) to artificial nucleases is indicated.     

DNA hydrolysis promoted by 1,7-dimethyl-1,4,7,10-tetraazacyclododecane

Several acyclic and macrocyclic polyamines were evaluated for their ability to cleave DNA. 1,7-Dimethyl-1,4,7,10-tetraazacyclododecane (DMC) could hydrolyze double-strand DNA at a concentration of 25microM. pH 7.2 was the optimal condition to cleave DNA in the presence of DMC. Supercoiled DNA hydrolytic cleavage by DMC was supported by the evidence from free radical quenching and T4 ligase ligation.     

Modulation of MutS ATP hydrolysis by DNA cofactors

Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics. At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM. Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly. These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.     

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn(2+) and Mn(2+) cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (4(4)) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N(40) DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA.     

Selective hydrolysis of damaged DNA by nuclease P1

The turnover rates for hydrolysis by nuclease P1 of the 16 unmodified dideoxynucleoside monophosphates were measured. In addition, the turnover rates were measured in a variety of dideoxynucleoside monophosphates containing free radical-induced base modifications. The modified bases included cis-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), 5,6-dihydrothymine, 5-hydroxymethyuracil, 8-hydroxyguanine, 5-hydroxy-5-methylhydantoin and the formamido remnant which can be derived from either a thymine or a cytosine base. The turnover rate for dinucleoside monophosphates containing 4,8-dihydro-4-hydroxy-8-oxo-guanine modifications, which are induced by singlet oxygen, were also measured. A model was devised for the hydrolysis of DNA by nuclease P1 which uses the observed turnover rates as parameters. The model predicts the abundance of monomers and dimers as hydrolysis proceeds. Whereas the level of monomers increases monotonically, the level of each dimer first increases and then falls off. There are advantages to phosphorylating dimers, as compared with monomers, using polynucleotide kinase. Consequently this model may be of interest in connection with ³²P-postlabeling applied to the measurement of DNA damage in nuclease P1 partial hydrolysates of DNA.     

The alkaline hydrolysis of phosphotriesters in alkylated mammalian DNA

Isolated DNA was alkylated with $\text{N-[}{}^{14}\text{C]methyl}\text{-N-}\text{nitrosourea}$ or $\text{N-[}{}^{14}\text{C]ethyl}\text{-N-}\text{nitrosourea}$. Sedimentation analysis of the alkylated DNA before and after alkaline hydrolysis was used to determine the number of single-strand breaks introduced by hydrolysis of the triesters. Vacuum distillation from alkylated DNA solutions before and after alkaline hydrolysis was used to determine the numbers of triesters hydrolysing to the alcohol.     

Stereochemical course of DNA hydrolysis by nuclease S1

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.     

A study of DNA depolymerisation during feulgen acid hydrolysis

Summary The binding of Schiff dye molecules after acid hydrolysis (1 M HCl) for varying lengths of time was studied in ascites tumour cells. The amount of dye bound to the tumour cells closely followed the number of aldehyde groups, calculated from the extraction of radioactive nucleotides. This constant dye to aldehyde ratio did not change when the hydrolysis was performed at a lower acid concentration (0.3 M HCl). The conclusion drawn is that Feulgen dye measurements represent, in a constant way, the number of aldehydes on DNA at any given time during hydrolysis. The alteration of the hydrolysis pattern of chromatin fixed in formalin was found to be due to a slower extraction of DNA depolymerisation products, the purine liberation being unaffected. A similar explanation is offered for the extreme pattern obtained from hydrolysis of bull spermatozoa chromatin.     

Catalytic hydrolysis of DNA by metal ions and complexes

Biomimetic hydrolysis of DNA or RNA is of increasing importance in biotechnology and medicine. Most natural nuclease enzymes that mediate such reactions utilize metal ion cofactors. Recent progress in the design of synthetic metallonucleases has included complexes of antibiotics, peptides, nucleic acids, and other organic ligands. In this article, we review a number of synthetic catalyst systems that have been developed to achieve efficient DNA hydrolysis. Methods to evaluate their catalytic efficiencies are critically discussed, and a prognosis for future work in this area is presented.     

DNA sequence dependence of ATP hydrolysis by RecA protein

The DNA sequence dependence of the ATPase activity of RecA protein has been investigated for a variety of single strand octamer and hexadecamer homopolymers and alternating copolymers. Under assay conditions where the single strand DNA concentration exceeds the RecA protein concentration, significant differences in the rates of ATP hydrolysis for the various single strand DNA oligomer cofactors are observed. Under the conditions examined, the order of efficiency of the DNA cofactors in inducing RecA mediated ATPase activity is found to be: dA16 greater than dT16 greater than d(TC)16 greater than dT8 greater than dC16 greater than dA8 = dG8 greater than dG16 greater than dC8 greater than d(AG)16. These results demonstrate not only a dependence of RecA ATPase activity on the sequence composition of short single strand DNA they further reveal ATPase activity can be affected by the nearest neighbor nucleotide sequence of short DNA cofactors.     

Locking the DNA gate of DNA gyrase: investigating the effects on DNA cleavage and ATP hydrolysis

Supercoiling by DNA gyrase involves the passage of one segment of double-stranded DNA through another. This requires a DNA duplex to be cleaved and the broken ends separated by at least 20 A. This is accomplished by the opening of a dimer interface, termed the DNA gate, which is covalently attached to the broken ends of the DNA. After strand passage, the DNA gate closes allowing the reunion of the broken ends. We have cross-linked the DNA gate of gyrase using cysteine cross-linking to block gate opening. We show that this locked gate mutant can bind quinolone drugs and perform DNA cleavage. However, locking the DNA gate prevents strand passage and the ability of DNA to stimulate ATP hydrolysis. We discuss the mechanistic implications of these results.     

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.     

Real-time direct observation of single-molecule DNA hydrolysis by exonuclease III

Single-molecule DNA digestion by exonuclease III, which has 3' to 5' exonuclease activity, was analyzed using a micro-channel with two-layer laminar flow. First, a DNA-bead complex was optically trapped in one layer in the absence of exonuclease III permitted the DNA to be stretched by the laminar flow. The exonuclease III reaction was initiated by moving the trapped DNA-bead complex to another layer of flow, which contained exonuclease III. As the reaction proceeded, the fluorescently-stained DNA was observed to shorten. The process was photographed; examination of the photographs showed that the DNA molecule shortened in a linear fashion with respect to the reaction time. The digestion rate obtained from the single-molecule experiment was compared to that measured from a bulk experiment and was found to be ca. 28 times higher than the bulk digestion rate.