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.     

Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study

Metallonucleases conduct metal-dependent nucleic acid hydrolysis. While metal ions serve in multiple mechanistic capacities in these enzymes, precisely how the attacking water is activated remains unclear for those lacking an obvious general base. All arguments hinge on appropriate pK_as for active site moieties very close to this species, and measurement of the pK_a of a specific water molecule is difficult to access experimentally. Here we describe a computational approach for exploring the local electrostatic influences on the water-derived nucleophile in metallonucleases featuring the common PD…(D/E)xK motif. We utilized UHBD to predict the pK_as of active site groups, including that of a water molecule positioned to act as a nucleophile. The pK_a of a Mg(II)-ligated water molecule hydrogen bonded to the conserved Lys70 in a Mg(II)-PvuII enzyme complex was calculated to be 6.5. The metal and the charge on the Lys group were removed in separate experiments; both resulted in the elevation of the pK_a of this water molecule, consistent with contributions from both moieties to lowering this pK_a. This behavior is preserved among other PD…(D/E)xK metallonucleases. pK_as extracted from the pH dependence of the single turnover rate constant are compared to previous experimental data and the above predicted pK_as.     

Synthetic metallonucleases for RNA cleavage

Synthetic metallonucleases are versatile metal ion catalysts that use multiple catalytic strategies for the cleavage of RNA. Recent work in the design of more active metallonucleases combines a single metal ion with functional groups that interact with RNA, including amino acid fragments or additional metal ions. Rate enhancements by multifunctional catalysts for cleavage of simple model substrates with good leaving groups are as high as 10(6) but somewhat lower (10(5)) for real RNA. However, cleavage of RNA substrates is complicated by different binding modes and steric interactions that can interfere with catalysis. Antisense oligonucleotides, peptides and small molecules that act as RNA recognition agents increase the strength of substrate binding, but not necessarily the catalytic rate constant. In general, catalytic strategies used by synthetic metallonucleases are probably not optimized. A better grasp of the mechanism of RNA cleavage by metal ions and more effort on positioning the metal ion complex with respect to the cleavage site may lead to improved catalysts.     

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.     

Non-hexameric DNA helicases and translocases: mechanisms and regulation

Helicases and nucleic acid translocases are motor proteins that have essential roles in nearly all aspects of nucleic acid metabolism, ranging from DNA replication to chromatin remodelling. Fuelled by the binding and hydrolysis of nucleoside triphosphates, helicases move along nucleic acid filaments and separate double-stranded DNA into their complementary single strands. Recent evidence indicates that the ability to simply translocate along single-stranded DNA is, in many cases, insufficient for helicase activity. For some of these enzymes, self assembly and/or interactions with accessory proteins seem to regulate their translocase and helicase activities.     

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.     

One- and two-metal ion catalysis: global single-turnover kinetic analysis of the PvuII endonuclease mechanism

Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal-dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single-turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of the metallonuclease mechanism. DNA association rate constants are approximately 100-fold higher in the presence of the catalytically nonsupportive Ca(II) versus the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM(2)S and EM(4)S, respectively) support cleavage. Interestingly, the cleavage rate for EM(2)S is approximately 100-fold slower than that displayed by EM(4)S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two-metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.     

Helicase motifs: the engine that powers DNA unwinding

Helicases play essential roles in nearly all DNA metabolic transactions and have been implicated in a variety of human genetic disorders. A hallmark of these enzymes is the existence of a set of highly conserved amino acid sequences termed the 'helicase motifs' that were hypothesized to be critical for helicase function. These motifs are shared by another group of enzymes involved in chromatin remodelling. Numerous structure-function studies, targeting highly conserved residues within the helicase motifs, have been instrumental in uncovering the functional significance of these regions. Recently, the results of these mutational studies were augmented by the solution of the three-dimensional crystal structure of three different helicases. The structural model for each helicase revealed that the conserved motifs are clustered together, forming a nucleotide-binding pocket and a portion of the nucleic acid binding site. This result is gratifying, as it is consistent with structure-function studies suggesting that all the conserved motifs are involved in the nucleotide hydrolysis reaction. Here, we review helicase structure-function studies in the light of the recent crystal structure reports. The current data support a model for helicase action in which the conserved motifs define an engine that powers the unwinding of duplex nucleic acids, using energy derived from nucleotide hydrolysis and conformational changes that allow the transduction of energy between the nucleotide and nucleic acid binding sites. In addition, this ATP-hydrolysing engine is apparently also associated with proteins involved in chromatin remodelling and provides the energy required to alter protein-DNA structure, rather than duplex DNA or RNA structure.     

Dissecting the metal ion dependence of DNA binding by PvuII endonuclease.

Divalent cations can provide an effective means of modulating the behavior of nucleic acid binding proteins. As a result, there is strong interest in understanding the role of metal ions in the function of both nucleic acid binding proteins and their enzymes. We have applied complementary fluorescence spectroscopic and nitrocellulose filter binding assays to quantitate the role of metal ions in mediating DNA binding and sequence specificity by the representative PvuII endonuclease. At pH 7.5 in the presence of the catalytically nonsupportive Ca(II), this enzyme binds the PvuII target sequence with a K(d) of 50 pM. Under strict metal-free conditions, the enzyme exhibits a K(d) of only 300 nM for the cognate sequence, an affinity which is weak relative to those measured for other systems in the absence of metal ions. This represents a 6000-fold increase in PvuII affinity for cognate DNA upon the addition of Ca(II). The pH dependences of both metal ion-dependent and metal ion-independent DNA binding are remarkably shallow throughout the physiological range; other characterized restriction enzymes exhibit more pronounced pH dependences of DNA binding even in the absence of metal ions. Similar measurements with noncognate sequences indicate that divalent metal ions are not important to nonspecific DNA binding; K(d) values are approximately equal to 200 nM throughout the physiological pH range, a behavior shared with other endonucleases. While some of these results extend somewhat the range of expected behavior for restriction enzymes, these results indicate that PvuII endonuclease shares with other characterized systems a mechanism by which cognate affinity and sequence discrimination are most effectively achieved in the presence of divalent metal ions.     

Multiple roles for ATP hydrolysis in nucleic acid modifying enzymes

Enzymes that operate on nucleic acid substrates are faced with the unusual situation where the substrate is much larger than themselves. Despite the potential to promote catalysis by utilizing the significant binding energy available through their interaction with substrate, ATP hydrolysis is frequently a part of the mechanism of these enzymes. The reasons for this have become clearer in recent years, and a surprising range of ways that these enzymes utilize the free energy of hydrolysis of ATP has been revealed. This review describes these different mechanisms in the context of the biochemical reactions that they support.     

Superfamily I helicases as modular components of DNA-processing machines

Helicases are a ubiquitous and abundant group of motor proteins that couple NTP binding and hydrolysis to processive unwinding of nucleic acids. By targeting this activity to a wide range of specific substrates, and by coupling it with other catalytic functionality, helicases fulfil diverse roles in virtually all aspects of nucleic acid metabolism. The present review takes a look back at our efforts to elucidate the molecular mechanisms of UvrD-like DNA helicases. Using these well-studied enzymes as examples, we also discuss how helicases are programmed by interactions with partner proteins to participate in specific cellular functions.     

In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme

A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the ‘8–17’ deoxyribozyme isolated under different conditions, including different design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribonucleotide base and G can be either ribo- or deoxyribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline earth metal ions. At saturating concentrations of Zn²⁺, the cleavage rate is 1.35 min^–1 at pH 6.0; based on pH profile this rate is estimated to be at least ~30 times faster at pH 7.5, where most assays of Mg²⁺-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes are capable of binding transition metal ions such as Zn²⁺ with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg²⁺-dependent nucleic acid enzymes under similar conditions.     

Understanding the transition states of phosphodiester bond cleavage: insights from heavy atom isotope effects

The nucleotides of DNA and RNA are joined by phosphodiester linkages whose synthesis and hydrolysis are catalyzed by numerous essential enzymes. Two prominent mechanisms have been proposed for RNA and protein enzyme catalyzed cleavage of phosphodiester bonds in RNA: (a) intramolecular nucleophilic attack by the 2'-hydroxyl group adjacent to the reactive phosphate; and (b) intermolecular nucleophilic attack by hydroxide, or other oxyanion. The general features of these two mechanisms have been established by physical organic chemical analyses; however, a more detailed understanding of the transition states of these reactions is emerging from recent kinetic isotope effect (KIE) studies. The recent data show interesting differences between the chemical mechanisms and transition state structures of the inter- and intramolecular reactions, as well as provide information on the impact of metal ion, acid, and base catalysis on these mechanisms. Importantly, recent nonenzymatic model studies show that interactions with divalent metal ions, an important feature of many phosphodiesterase active sites, can influence both the mechanism and transition state structure of nonenzymatic phosphodiester cleavage. Such detailed investigations are important because they mimic catalytic strategies employed by both RNA and protein phosphodiesterases, and so set the stage for explorations of enzyme-catalyzed transition states. Application of KIE analyses for this class of enzymes is just beginning, and several important technical challenges remain to be overcome. Nonetheless, such studies hold great promise since they will provide novel insights into the role of metal ions and other active site interactions.     

The role of metal ions in RNA catalysis

Understanding the catalytic mechanisms of RNA enzymes remains an important and intriguing challenge - one that has grown in importance since the recent demonstration that the ribosome is a ribozyme. At first, it seemed that all RNA enzymes compensate for the limited chemical versatility of ribonucleotide functional groups by recruiting obligatory metal ion cofactors to carry out catalytic chemistry. Mechanistic studies of the large self-splicing and pre-tRNA-processing ribozymes continue to support this idea, yielding increasingly detailed views of RNA active sites as scaffolds for positioning catalytic metal ions. Re-evaluation of the methodologies used to distinguish catalytic and structural roles for metal ions, however, has challenged this notion in the case of the small self-cleaving RNAs. Recent studies of the small ribozymes blur the distinction between catalytic and structural roles for metal ions, and suggest that RNA nucleobases have a previously unrecognized capacity for mediating catalytic chemistry.     

Mapping metal ions at the catalytic centres of two intron-encoded endonucleases.

Divalent metal ions play a crucial role in forming the catalytic centres of DNA endonucleases. Substitution of Mg2+ ions by Fe2+ ions in two archaeal intron-encoded homing endonucleases, I-DmoI and I-PorI, yielded functional enzymes and enabled the generation of reactive hydroxyl radicals within the metal ion binding sites. Specific hydroxyl radical-induced cleavage was observed within, and immediately after, two conserved LAGLIDADG motifs in both proteins and at sites at, and near, the scissile phosphates of the corresponding DNA substrates. Titration of Fe2+-containing protein-DNA complexes with Ca2+ ions, which are unable to support endonucleolytic activity, was performed to distinguish between the individual metal ions in the complex. Mutations of single amino acids in this region impaired catalytic activity and caused the preferential loss of a subset of hydroxyl radical cleavages in both the protein and the DNA substrate, suggesting an active role in metal ion coordination for these amino acids. The data indicate that the endonucleases cleave their DNA substrates as monomeric enzymes, and contain a minimum of four divalent metal ions located at or near the catalytic centres of each endonuclease. The metal ions involved in cleaving the coding and the non-coding strand are positioned immediately after the N- and C-terminally located LAGLIDADG motifs, respectively. The dual protein/nucleic acid footprinting approach described here is generally applicable to other protein-nucleic acid complexes when the natural metal ion can be replaced by Fe2+.     

Kinetic analysis of product release and metal ions in a metallonuclease

Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equilibrium and kinetic experiments are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equilibrium fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre-steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre-steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, analysis of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no positive cooperativity between the two metal ions binding each active site.