The catalytic mechanism of DNA and RNA polymerases - ACS Catalysis

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Perspective

The catalytic mechanism of DNA and RNA polymerases Vito Genna, Elisa Donati, and Marco De Vivo ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b03363 • Publication Date (Web): 18 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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ACS Catalysis

The Catalytic Mechanism of DNA and RNA Polymerases Vito Genna,1 Elisa Donati1 and Marco De Vivo1

1. Laboratory of Molecular Modeling and Drug Discovery, Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genoa, Italy

Corresponding author: Dr. Marco De Vivo Email: [email protected] Phone: +39 010 71781577

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Abstract. DNA and RNA polymerases (Pols) catalyze nucleic acid biosynthesis in all domains of life, with implications for human diseases and health. Pols carry out nucleic acid extension through the addition of one incoming nucleotide trisphosphate at the 3’-OH terminus of the growing primer strand, at every catalytic cycle. Thus, Pol catalysis involves chemical reactions for nucleophile 3’OH deprotonation and nucleotide addition, as well as major protein conformational motions and structural rearrangements for nucleotide selection, binding, and nucleic acid translocation to complete the overall catalytic cycle. In this respect, quantum and molecular mechanics simulations, integrated with experimental data, have advanced our mechanistic understanding of how Pols operate at the atomic level. This Perspective outlines how modern molecular simulations can further deepen our understanding of Pol catalytic reactions and fidelity, which may help in devising strategies for designing drugs and artificial Pols for biotechnological and clinical purposes.

Keywords: DNA, RNA, Polymerases, Catalysis, QM/MM, Molecular Mechanics, Computations, Modeling


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Introduction DNA and RNA polymerases (Pols) are enzymes that play a major role in crucial cellular processes, such as gene expression, regulation, transcription, and repair.1-3 As such, Pols are critical pharmacological targets for treating a wide range of diseases including viral and bacterial infection, neurodegenerative diseases, and cancer.4-6 In recent decades, this has prompted extensive efforts to better understand their mechanism of action.7-10 Indeed, an improved understanding of Pol catalysis has led to new drug discovery strategies for modulating Pol function, and to the engineering of innovative artificial Pols for biotechnological and therapeutic purposes.11-14 Pols elongate the nascent nucleic acid strand via a complex stepwise catalytic process with both chemical and physical steps. First, during each catalytic cycle, Pols extend the growing primer

strand

by

adding

one

incoming

nucleotide

at

the

3′

primer

terminus

(deoxyribonucleoside triphosphate, dNTP, for DNA Pols; ribonucleoside triphosphate, rNTP, for RNA Pols). This occurs via a well-characterized SN2-like phosphoryl-transfer reaction, which is assisted by the conserved two-metal-ion mechanism (typically involving Mg or Mn), as demonstrated by a wealth of structural data on ternary Pol/(R)DNA/(d)NTP complexes.15-17 Thus, Pol catalysis comprises chemical reactions for primer 3’-OH deprotonation (i.e. nucleophile activation) and nucleotide addition, which allow the nascent strand to be elongated, one incoming nucleotide at a time (Fig. 1). As part of the same catalytic cycle, Pols undergo functional large-amplitude protein conformational motions18-20 and structural rearrangements for nucleotide selection,21,

22

binding,23-25 and nucleic acid translocation.26-29 Together, these

chemical and physical steps constitute the overall catalytic cycle for processive nucleic acid biosynthesis catalyzed by Pols (Fig. 1). In this context, the rapidly growing body of Pol experimental data has revealed many key aspects of Pol catalysis.30 In particular, time-resolved X-ray crystallography has enabled researchers to capture and observe the chemical reaction for nucleotide addition, moving from the reactants to the products.31-33 One such example is the recent in crystallo catalysis observed


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for human DNA Pol- a eukaryotic Pol involved in DNA repair and recombination34, 35 (Fig. 2). These structural data have generated important atomistic insights into (d)NTP incorporation, which occurs via the nucleophilic attack of the primer terminal 3′-OH to the α-phosphorus of the incoming dNTP.34 That is, the phosphoryl-transfer reaction for nucleotide addition is catalyzed via the established two-metal-ion mechanism, where two Mg ions facilitate nucleophile formation, stabilize the transition state, and help the leaving group departure.36-39 Indeed, passing from the reactants to the products, Pol-’ crystals clearly show the typical inversion of the umbrella of the scissile phosphate during the phosphoryl-transfer reaction, as expected for a canonical SN2-like reaction.38 This leads to the formation of the pyrophosphate (PPi) product and the consequent conclusion of the catalytic cycle for nucleotide addition (Fig. 1).34,

40

However, the transient nature of the enzymatic transition state poses technical

limitations in determining the exact spatiotemporal location and structural features of this highenergy state. Moreover, there is debate over the mechanism and possible pathways of protontransfer events during catalysis, such as the one needed to deprotonate and activate the nucleophilic -3′O atom of the primer.41-45 In addition, recent ternary Pol/D(R)NA/d(r)NTP complexes have revealed the presence of an extra metal ion transiently bound to the catalytic site (Table 1).46, 47 This third cation has been observed in a few Pols structures, including in some Pol- It seems to be an additional key player for Pol catalysis, although its exact role is unclear. A few recent and comprehensive reviews have mostly focused on the structural and kinetics data of Pol enzymes.48-50 As mentioned above, in addition to the chemical steps for nucleotide incorporation, Pol function requires key physical steps for the nucleotide selection, binding, and nucleic acid translocation to complete the catalytic turnover. Importantly, these events also regulate Pol fidelity, which is the ability of Pols to faithfully replicate the template strand during catalysis. Pol fidelity requires the contribution of protein dynamical features for correct nucleotide recognition and binding.51-53 This occurs prior to nucleotide incorporation into the primer strand, with subsequent formation of the canonical Watson-Crick base pair.54 Yet the underlying molecular mechanism for Pol fidelity remains uncertain. Furthermore, for highly processive ACS Paragon Plus Environment

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DNA/RNA synthesis defined by recurrent catalytic cycles for nucleotide addition, the Pols must efficiently translocate along the nucleic acid strand by one base pair. This catalytic step again requires large conformational rearrangements of the Pol-substrate complex. In this regard, the abundance of structural and kinetics data on Pol-, a small eukaryotic Pol for DNA repair,55 has greatly helped in understanding how Pol fidelity is enabled by major conformational changes during the overall catalytic cycle.56-58 In addition, nucleic acid translocation also requires the efficiency and coordination of large-amplitude motions of structural regions of the ternary Pol/D(R)NA/(d)NTP complex. This relates to Pol’s processivity, i.e. the number of incoming nucleotides incorporated along the growing primer strand during a single template-binding episode, before the Pol enzyme dissociates from the nucleic acid strand.15 Insights into these chemical and physical steps for Pol-assisted nucleic acid biosynthesis are critical to better understanding Pol catalysis. In this respect, quantum and molecular mechanics simulations have helped advance our atomistic understanding of Pol function.59-63 This Perspective analyzes the existing mechanistic hypotheses concerning Pol catalysis and fidelity in light of recent structural data and computational studies on Pol function. We conclude by examining artificial Pol enzymes, which have the potential to impact positively on human health. In sum, we outline how state-of-the-art multiscale molecular dynamics simulations, combined with experimental data, will help to deepen our understanding of nucleic acid synthesis performed by DNA/RNA Pols.

An intramolecular H-bond characterizes the typical geometry of the incoming nucleotide. To be added to the growing nucleic acid strand, the incoming nucleotide must enter into and bind to the two-metal-aided catalytic site of Pols (Fig. 1). Once bound to the (R)DNA-Pol binary complex, the conformation adopted by the nucleotide allows the SN2-like phosphoryl-transfer reaction for nucleotide addition, according to the two-metal-ion mechanism.36, 38 However, the preferred molecular conformation of nucleotides in water is radically different from the conformation adopted by nucleotides in ternary Pol/D(R)NA/(d)NTP complexes. Indeed, when freely dispersed in solution, the (d)NTP has been reported to prefer extended and relaxed ACS Paragon Plus Environment

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conformations, in which the triphosphate is bound to one divalent metal ion.64 This has been demonstrated by computational studies that used quantum mechanics/molecular mechanics (QM/MM) simulations to elucidate the mechanisms of ATP hydrolysis in water.65, 66 However, when the nucleotide is bound to the binary (R)DNA-Pol complex, it always shows a much more compacted conformation. This conformation is characterized by an intramolecular and stereospecific H-bond formed by the nucleophilic 3′-OH with the pro-S oxygen atom of the phosphate of the incoming nucleotide (Fig. 2). Notably, we recently demonstrated that this conformation is consistently preserved in all the nucleotides co-crystallized in ternary Pol/(R)DNA/(d)NTP complexes from all domains of life (Fig. 3A).41 In contrast to linear conformations observed in water, this typical (d)NTP architecture allows the incoming nucleotide to form base stacking with the preceding nucleobase along the nascent primer strand, while also forming base pairing with the templating nucleotide. Concomitantly, this conformation allows the proper placement of the (d)NTP -phosphate on top of the two metal ions within the catalytic site. In this way, the alkoxide nucleophilic species can now attack Pα thanks to the collinearity between the 3’O- group and the oxygen atom bridging the Pα and Pβ atoms, as expected for the canonical SN2-type reaction scheme.67-73 Therefore, it seems that a prerequisite for catalysis is the conformational change of the incoming nucleotide, which must move from an extended conformation in water to a much more bent geometry when in the catalytic pocket. The intramolecular H-bond helps in forming and maintaining this conformation. In addition, we have noted that this short H-bond co-exists with the reactive C3′-endo conformation of the sugar pucker adopted in the Michaelis-Menten complex by the incoming nucleobase (Fig. 3B).41 We have used these structural observations to propose a self-activated mechanism (SAM) for nucleic acid elongation catalyzed by Pols (vide infra). More recently, Wu et al. commented on the relevance and possible role of this conserved intramolecular H-bond.50 In particular, they observed that 2ʹ,3ʹ-dideoxy-NTP (ddNTP), which is usually a chain terminator in DNA sequencing, can be incorporated into DNA by polymerases even if it lacks hydroxyl groups on the sugar ring. That is, ddNTP cannot form such an intramolecular H-bond. This could explain ACS Paragon Plus Environment

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the observed >100-fold reduction of the catalytic efficiency for ddNTP incorporation by Pols.50 Together, these indications suggest that this structurally conserved H-bond established between the nucleophilic 3′-OH and the -phosphate of the incoming nucleotide may indeed be functional for catalytic nucleotide addition. Intuitively, this conformational change of the nucleobase with intramolecular H-bond formation likely occurs during nucleotide recognition and binding. However, the energetics, dynamics, and spatiotemporal occurrence of this conformational rearrangement must still be elucidated.

Nucleophile formation and nucleotide addition for nucleic acid elongation. Pol-mediated nucleic acid elongation requires two chemical steps, i.e. nucleophile formation and subsequent nucleophilic attack to incorporate the incoming nucleotide into the growing primer strand, with consequent formation of the pyrophosphate (PPi) leaving group.38,

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Over the years,

computational enzymology has generated and analyzed a few proposals for the mechanism used by Pols to perform nucleotide incorporation at the 3′ primer terminus, in a 5’→3’ direction. There are already comprehensive reviews and numerous experimental studies of the kinetics and structural data related to Pol catalysis.30,

50, 74, 75

Here, we focus on how computational

methods have been used to investigate the enzymatic mechanisms of Pol-mediated nucleic acid biosynthesis. A first mechanistic hypothesis was proposed by Warshel and coworkers, who investigated the catalytic mechanism for nucleotide incorporation by the DNA Pol of bacteriophage T7.44 In this mechanistic study, the nucleophilic oxygen is preferentially activated by a nearby aspartate (Asp654), when compared to mechanisms for nucleophile activation involving, as proton acceptor, either bulk water or one of the oxygens of the -phosphate of the incoming nucleobase (Fig. 4). Thus, the favored mechanism was the one where the ionized Asp654 acts as a general base, receiving the proton from the attacking 3′-OH of the primer strand. An analogous deprotonation mechanism was also proposed for DNA Pol-, with QM/MM calculations used to propose that an aspartate residue (Asp256) is the general base for activating the terminal primer deoxyribose -O3′.76 The same mechanism was also reported in ACS Paragon Plus Environment

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DNA Pol-λ, where computation supports that an aspartate (Asp490) acts as proton acceptor for nucleophile activation.77 A final example is a more recent computational study of the catalytic mechanism of the DNA Pol-I. This study proposed that a histidine residue (His829), instead of an aspartate, may accept the proton of the attacking 3′-OH, and thus activate the nucleophile.78 The common mechanistic feature of these proposals for nucleophile activation is the presence of a first coordination-shell residue that can act as a general base (i.e. proton acceptor) for the deprotonation of the attacking oxygen.78 Following this first protein-mediated chemical step for nucleophile activation, the reaction proceeds according to the two-metal-ion mechanism for nucleophilic attack on the -phosphate of the dNTP substrate, and departure of the PPi leaving group. An alternative mechanism for nucleophile formation and nucleotide addition was later proposed by Zhang and coworkers, who hypothesized that deprotonation of the attacking 3′OH of the primer strand may occur via a nearby water, which can act as a proton acceptor (Fig. 4).43 Eventually, this water shuttles the proton on the leaving PPi group. This is referred to as a water-mediated-substrate-assisted (WMSA) mechanism. It was proposed and examined via QM/MM simulations of the lesion-bypass DNA polymerase IV (Dpo4) catalysis for a nucleotidyl transfer reaction. The same research group also described the WMSA mechanism for the T7 DNA Pol42 and DNA Pol- enzymes.79 Later, Wang et al. reported and computationally examined a similar water-mediated mechanism for nucleophile formation in DNA polymerase IV80 (Dpo4) catalysis, during which two water molecules facilitate the initial deprotonation of the primer 3′OH, then the shuttling of the migrating proton to the leaving group. In the case of RNA polymerase II (RNA Pol-II), one hydroxide ion from the bulk was found to be the favored proton acceptor for nucleophile activation.81 Other possible pathways to shuttle the migrating proton to the PPi group were also proposed.81, 82 The PPi is then released via motions of a specific loop,83 as depicted via a Markov state model (MSM) constructed from extensive all-atom molecular dynamics (MD) simulations.84, 85 A final mechanistic scheme for Pol catalysis, which differs from the above hypotheses, is our recent proposal for a self-activated mechanism (SAM).41 It is ‘self-activated’ because the ACS Paragon Plus Environment

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mechanism is initiated by the intramolecular H-bond found in the incoming nucleotide (as described above, Figs. 3 and 5). In SAM, this intramolecular H-bond prompts the in situ deprotonation of the 3′-OH in the incoming nucleotide. Our Car-Parrinello (CP) QM/MM simulations have shown that the proton of the 3′-OH of the entering nucleotide can be directly transferred onto the pro-S oxygen of the -phosphate as the nucleotide addition occurs. That is, the proton transfer occurs simultaneously with the detachment of the -phosphate from the -phosphate of the incoming nucleobase, leading to the formation and departure of the leaving (and protonated) PPi group (Fig. 5). Notably, in SAM, the newly formed 3’-hydroxide ion in the incoming nucleotide is therefore formed and located on top of MgA during nucleic acid translocation. In this way, the deprotonated 3’-hydroxide is ready for a new catalytic cycle for nucleotide addition. This mechanism for the deprotonation of the 3′-OH of the incoming nucleobase during nucleotide addition is one difference between SAM and the previously proposed mechanisms for Pol catalysis. However, in SAM, the nucleophile is formed as nucleotide addition occurs thanks to the activated 3’-hydroxide ion of the attacking group at the primer strand (formed via the same intramolecular proton transfer, during the previous catalytic cycle). Importantly, this key element of SAM allows the synergistic interconnection of a concerted closed-loop catalytic sequence of events that include the chemical steps for nucleophile deprotonation and nucleotide addition (as in the previous mechanistic proposals), and also the physical step for nucleic acid translocation (not considered in the previous mechanistic proposals). In this regard, at least in our case, ab initio QM/MM simulations (as opposed to static calculations) were instrumental in characterizing this concerted structural rearrangement associated to nucleophile activation and leaving group formation that occur simultaneously with, notably, partial nucleic acid translocation for Pol catalysis. Thus, the coupling between the chemical and physical steps, which form a closed-loop catalytic cycle for Pol catalysis, represents the novel conceptual aspect in SAM. However, the level of synchronicity and synergy of these concerted chemical and physical steps in SAM remains to be elucidated. As an aside, it is interesting that such concerted metal-aided mechanisms for proton transfer in biological ACS Paragon Plus Environment

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systems are somehow reminiscent of concepts used for homogeneous catalysis, with computationally characterized reaction mechanisms such as the “Concerted MetalationDeprotonation” (CMD) and “Ambiphilic Metal−Ligand Assistance” (AMLA) for carboxylateassisted C−H activation in organometallic systems.86-88

Different enzymatic mechanisms for the chemical steps in Pol activity. There are a few mechanistic hypotheses for Pol catalysis (see paragraph above), which are all equally plausible in principle (Fig. 4 and 5).41, 43, 44, 89 One main difference is the way the nucleophile is activated. Conveniently, one could argue that the chemical step for nucleophile activation is unlikely to affect the overall energetics of nucleotide addition. If so, one could start investigating the enzymatic reaction from an already activated nucleophile that was somehow deprotonated.89 However, even if that were the case, nucleophile activation is undoubtedly needed for catalysis to be initiated and for it to proceed with the subsequent chemical and physical steps. It is therefore informative to investigate and understand how this key event for nucleophile deprotonation occurs at the atomic level. In all the possible mechanisms discussed above, MgA facilitates 3′-OH deprotonation by lowering its pKa, thus favoring the ionized 3′-O- form. Concomitantly, MgB facilitates leaving group formation.40 Once activated, the 3’-hydroxide ion can perform the subsequent nucleophile attack at the P of the incoming nucleotide, with a dissociative or associative transition state (TS, defined in this case as a metastable pentacovalent phosphorane species) for metal-aided nucleotidyl transfer reaction supported by MgA and MgB, which act together to stabilize the TS along the reaction path.36 In light of the extensive literature on enzymatic metal-aided phosphoryl transfer reactions (see e.g. Ref.68, 69, 90, 91), the precise geometry of the TS for phosphoryl transfer is expected to depend on the specific Pol enzyme under investigation. Any of the mechanisms described above for Pol catalysis may be valid for a subset of Pol enzymes. It would be a stretch to generalize and present a specific TS geometry (associative vs. dissociative) as a template for all Pol catalytic mechanisms. In all this, there is still debate over how to experimentally test the mechanistic details of Pol catalysis. The energetics of the reactions can be compared to the ACS Paragon Plus Environment

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experimental kcat using transition state theory. This comparison is commonly used to indirectly validate the mechanistic hypothesis.19,

92-94

Analogously, the computed energetics of the

enzyme-catalyzed reaction should be compared and validated by calculating the energetic barrier for the same uncatalyzed reaction in water, which is expected to be much higher.95-99 However, it is extremely difficult to experimentally demonstrate the specific pathways for nucleophile activation and proton migration, which lead the proton of the nucleophilic 3′-OH group to the final acceptor (bulk water, a protein residue, or the PPi leaving group). In this regard, for example, solvent deuterium isotope effects indicated two proton-transfer events for the RNA-dependent RNA polymerase (RdRp) from poliovirus, although their exact migration path is challenging to determine experimentally.100 However, such experimental studies are to be welcomed, because these are critical in order to examine and possibly verify mechanistic proposals about the pathways for proton migration, detected by computational investigations.

A third transient metal ion for nucleic acid biosynthesis. A recent new player in the twometal-ion mechanism for nucleic acid processing is an additional third metal ion (MgC), which was recently resolved close to and above MgB in Pol- structures.34, 47 Notably, a third solventexposed cation was provocatively proposed a few years ago as an active part of the two-metalion mechanism in ribonuclease H (RNase-H), based on evidence from MD simulations.101 It was recently found in a few structures of the human exonuclease 1 (hExo-1)102 and D. mobilis homing endonuclease (I-DmoI).103 Second-shell basic amino acids and cations have also been experimentally identified and computationally examined at structurally conserved positions in a large set of two-metal-ion enzymes that process DNA and RNA (including several Pols, nucleases and also ribozymes, Fig. 6), as well as metallolyase enzymes.103-108 Taken together, these experimental and computational results further support the idea that additional structural elements, located in a larger orbit of the metal-centered structural architecture, may be critical for the proper function of the two-metal-ion mechanism for nucleic-acid processing.104 In more detail, the third ion found in Pol- is precisely located within the enzymatic active site, on the opposite side of the 3’-OH group, and it chelates oxygens of the - and ACS Paragon Plus Environment

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phosphates of the dNTP. We have collected the available Pols structures resolved in the presence of the third ion (see Tab. 1). In addition to Pol-, there are structures from Pol-  and Pol-  enzymes, including the engineered Pol Kod-RI,111 an artificial threose nucleic acid (TNA) polymerase.112 Notably, in Kod-RI111 (and in Pol-,113 the third metal ion has a different location than in native Pols. Intriguingly, however, it is still in direct contact with the entering nucleotide. In all these structures, the catalytic metal ions are most often Mg and Mn, although Ca and Na are sometimes found in these structures, according to the specific conditions used to obtain the crystals (see Tab. 1). The surprising finding of a third metal ion in Pols has opened a stimulating and unresolved debate about the specific role of this additional ion for Pol catalysis.46 While the third metal ion seems functional,46, 114 there are at least three hypotheses about its mechanistic role, which are not necessarily mutually exclusive. Indeed, examining the structures containing this third ion (such as the MgC captured in Pol- during in crystallo reactions via time-resolved X-ray crystallography),47 it is interesting that this ion is mostly present bound in structures resembling the TS for nucleotide addition, or just after the chemical reaction occurred.34 Indeed, the diffusion and protein binding of this third ion is expected to occur on a timescale longer than that of the chemical reaction for nucleotide addition. It is thus unclear if metal binding indicates that this ion participates actively to the chemical reaction, stabilizes the products and/or facilitates group leaving departure. That is, the observed position of this transient third metal ion, and the time it appears along the reaction path resolved in crystallo, suggests that the third ion: i) may be necessary for the reaction to occur, further stabilizing the negative charge of the scissile phosphate during the nucleotidyl transfer, and/or ii) favors products formation, inhibiting the backward reaction from the products to the reagents, and/or iii) acts as an exit shuttle for the leaving PPi departure. In principle, these three different active roles for MgC during catalysis could be complementary or, at least, not necessarily unconnected. It is therefore challenging to ascertain whether the third ion performs one or more of those actions during catalysis.


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Yang and collaborators maintain that the third ion is essential for nucleotidyl transfer in Pols,47 as suggested by the fact that a low metal ion concentration in experiments for reaction in crystallo hampers catalysis, i.e. the product is not experimentally detected in crystallo if a low metal ion concentration is used.46 This experimental observation supports the active role of the third ion in lowering the energetic barrier for the enzymatic phosphoryl-transfer in Pols, thus accelerating dNTP hydrolysis. However, the fact that low metal ion concentration does not lead to product detection in crystallo does not necessarily exclude the contribution of MgC in stabilizing the products when the metal is at physiological concentrations. Likewise, it remains plausible that the third ion plays a role in facilitating leaving group departure by acting as an exit shuttle. Indeed, the third ion appears to prevent the reverse enzymatic reaction (i.e. pyrophosphorolysis) by stabilizing the products. This has been demonstrated by QM/MM computations from Wilson and coworkers115 on the role of the third ion in Pol-, and by our CP QM/MM simulations of Pol-.41 More recently, Yoon and Warshel used empirical valence-bond (EVB) calculations on Pol- structures116 to obtain computed energy profiles that confirm the role of the third ion in stabilizing the reaction products. This was further confirmed via QM/MM simulations by Stevens and Hammes-Schiffer.114 In addition to these studies, we have previously reported forcefield-based MD simulations that describe the role of the third ion in lowering the barrier for PPi release, acting as the exit shuttle.21 In this regard, the idea that a third ion is needed for Pol catalysis is challenged by recent in crystallo reactions and new structural data, together with primer extension assays reported by Kottur and Nair for DNA polymerase IV (PolIV).117 These results support the hypothesis that the PPi group is hydrolyzed after nucleotidyl transfer. This energetically favors DNA synthesis via the conventional two-metal-ion mechanism, at least in the case of Pol IV.117 Based on the available structural,47 kinetics,48 and computational evidence,114,

118

it is

therefore tempting to believe that the third ion, characterized by its transient nature, actively participates in the chemical step for nucleotide incorporation in Pols.34 In addition, a few recent computational studies from different groups suggest that the third ion stabilizes product formation, and acts as an exit shuttle to facilitate the departure of the leaving PPi outside of the ACS Paragon Plus Environment

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catalytic site.21, 28, 82, 119 Certainly, this puzzling and novel aspect of the common two-metalmechanism in Pols merits future experimental and computational studies to further elucidate whether and how a third metal ion is an active player in this complex and fascinating biological process for nucleic acid biosynthesis.

Insights into what regulates fidelity in Pols. The fidelity of Pol enzymes is expressed as the error rate per nucleotide that is added during template-strand replication.120,

121

While the

chemical steps of nucleophile deprotonation and nucleotide addition for Pol-mediated nucleotidyl transfer are rather spatially restricted to the region of the catalytic site, fidelity also relates to physical steps for nucleotide selection, recognition, and binding, which necessarily involve major protein conformational motions and structural rearrangements. For example, this has been experimentally determined for the Dpo4 and Pol- enzymes, as well as for several other Pols.48, 51, 122-124 Each Pol is able to recognize the correct nucleobase in solution, which is selectively recruited and inserted along the primer strand, complementing the template base.125-127 These structural changes must eventually involve nucleic acid translocation to allow one nucleotide to be added to the growing strand at each catalytic cycle.84, 128-130 In addition, fidelity and processivity (i.e. the number of incoming nucleotides added to the primer strand for each single template-binding event) can be modulated by the proofreading 3’→5’ exonuclease. If there is incorrect incorporation, specialized exonucleases can resolve mispairing by removing the incorrect nucleotide, which is cleaved from the end of the strand one at a time.131-133 This is also true for highly processive Pols, like Pol- and Pol- in eukaryotes, and Pol-I, Pol-II and Pol-III in prokaryotes.132, 134-136 Once this correction is performed, the nucleic acid chain can be transferred back into the Pol enzyme to restart the polymerization process. Overall, this intricate yet elegant sequence of chemical and physical events defines Pol fidelity and processivity. In terms of computational studies, much effort has been devoted to examining the structural features and energetics of dynamical transformations and reactions that may contribute to fidelity and processivity in Pols. For example, a few studies have analyzed how Pols are able to ACS Paragon Plus Environment

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avoid the addition of an incorrect nucleotide, if accidentally accommodated within the active site. One example is the MD-based investigation of fidelity of the T7 DNA Pol, where incorrect nucleotide binding generated a higher energetic barrier.19, 137 However, it remains unclear how this energy increment for incorrect nucleotide binding affects the energy for the subsequent nucleotide incorporation. In principle, one can hypothesize that the formation of a poorly reactive ternary Pol/(R)DNA/(d)NTP complex increases the energy barrier for nucleotide incorporation and, consequently, the chance for the incorrect nucleotide to dissociate from the Pol catalysis site, thus allowing binding of the correct nucleoside triphosphate.138 Moreover, the translocation step is critical for high-processivity, as shown by MD simulations of the DNA Pol-I, where this process is mediated by significant structural rearrangements regulated by conserved Pol residues.28,

139-141

All these events may also involve additional conserved second-shell

residues that have recently been reported to expand the two-metal-ion architecture in several nucleic-acid-processing enzymes.104 Then there are low-processive and low-fidelity Pols, such as Pol- which promotes misincorporation events in order to produce genome variability for antibody generation (a process named somatic hypermutation).35, 142-144 One hypothesis about these Pols is that the correct dNTP substrate remains preferentially bound to the binary Pol/dsDNA complex due to a more stable and better structured hydrogen bond pattern and base-stacking interaction.145 In particular, mutagenesis and kinetics studies have shown that the residue Arg61 in Pol- is critical for catalysis.146-148 The catalytic role of this specific residue was also confirmed via MD and QM/MM calculations, which revealed its contribution to stabilizing the TS for nucleotide addition, and facilitating PPi departure, acting in cooperation with MgC.21, 41 Forcefield-based MD simulations have shown that, compared to undamaged DNA, defined hydrogen bond patterns and stacking interaction favor binding of selective dNTPs to Pol- in the presence of DNA damage (thymine−thymine dimer, TTD).22, 149 The conserved Arg61 thus seems to affect Pol- fidelity and processivity. This is in line with experimental observations of this specific Pol.22, 149

This interpretation is also supported by other studies, which indicate that a specific enzyme

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fidelity Pols, such as Pol- and in T7-RNA Pol.128 A large dataset of Pol/DNA structures feature a positively charged residue (Lys or Arg) near the enzymatic two-metal active site.45, 151 In these structures, this conserved residue always interacts directly with the incoming nucleotide (Fig. 2). Recently, classical MD simulations and free-energy calculations152 have suggested that the Arg61-mediated stabilization of the incoming dNTP may favor the formation of the Watson−Crick (W−C) pairing in Pol- Fig. 7).151 Incidentally, distorted W−C or even Hoogsteen base-pairing conformations have been observed in some structures of low-fidelity human DNA Pol- and the African swine fever virus Pol-X.154,

155

In these structures, the

conserved positively charged residue (e.g. Arg61 in Pol- was either missing or slightly displaced.151 This positively charged residue is undoubtedly not the only element shaping the nascent nucleic acid geometry in Pols. Other structural elements may include recently identified and conserved second-shell residues.104 ‘Pre-chemistry protein conformational changes’ is the collective term for the catalytic sidechain rearrangements and/or major enzyme domain dynamics that occur prior to the catalytic step. The potential impact of pre-chemistry protein conformational changes on Pol catalysis is the subject of lively debate, with studies centered on Pol .20, 156-162 As thoroughly discussed by Mulholland et al.,157 the mechanistic implications of pre-chemistry conformational changes for Pol function depend on whether one considers protein dynamics in relation to the formation of a reactive protein conformation, or in terms of local atomic motions that may affect the reaction rate. Moreover, these arguments rely on specific assumptions about the rate-limiting step for Pol’s fidelity and reactivity, in the context of the complex kinetic behavior of the enzymatic system.157,163 In summary, we have described a stepwise and complex sequence of chemical and physical steps, which regulate nucleotide addition for nucleic acid elongation, and the fidelity and processivity of Pols in catalyzing nucleic acid biosynthesis. These steps include chemical reactions for the addition of a nucleotide to the growing primer, as well as large-amplitude conformational changes for substrate binding and unbinding, and the final nucleic acid translocation (or Pol dissociation from the template strand). But what is the rate-determining ACS Paragon Plus Environment

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step for this multifactorial DNA/RNA extension process? This question is the subject of intense investigation and debate. One proposition is that the rate-determining step may be different for different Pols, and may vary according to whether the inserted nucleotide is correct or not.154, 164-167

Indeed, correct and incorrect nucleotide incorporation may involve different

conformational changes for the recognition and binding of the entering nucleotide in every given Pol enzyme (thus affecting its fidelity).121, 126, 168, 169 A few cases have been reported where the physical step for pre-chemistry conformational changes is suggested to be rate-liming for Pol function.18, 170-171 However, based also on computational studies,28, 137, 172, 173, 160 another valid hypothesis is that the chemical step for nucleotide addition is the actual rate-liming step for Pol-mediated nucleic acid biosynthesis.15,

46, 174, 175

To further complicate the issue, it is

arduous to assess consistently the energy contribution of each discrete chemical and physical step for Pol catalysis and fidelity, relative to the energetic and kinetics of the overall Pols catalytic cycle. While there is no general consensus on the rate-limiting step, a thorough analysis and discussion can be found in Raper et al.’s comprehensive review of the kinetics mechanisms of Pols.48 In this respect, computational simulations will continue to play a critical role in interpreting and enriching the kinetics and structural data on Pol catalysis.

Outlook and conclusions. Pol enzymes play a crucial role in the synthesis and processing of nucleic acids within cells. For this reason, their overall functioning and catalysis are the subject of intense research. Pols are essential for biotechnology applications including DNA amplification, sequencing, and polymerization of synthetic genetic polymers.176-178 In addition, Pols are often targeted for therapeutic intervention, especially in cancer.59, 179-183 It is therefore critical to understand their mechanism in order to modulate their function. Here, we have outlined how computation can help to elucidate the enzymatic mechanism of Pol enzymes. More generally, a computational researcher can nowadays choose from among many different codes and methods based on different levels of theory (e.g. forcefield-based, semi-


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empirical like in EVB, ab initio, DFT and even more costly/accurate quantum calculations).184-194 These can be coupled to one or more approaches for free-energy calculations (e.g. thermodynamic integration, metadynamics, umbrella sampling, free-energy perturbation, transition-path sampling, minimum free-energy path scheme, string methods, and others).60, 195-200

As we have shown, these different computational methods and approaches can be used

to interrogate enzymatic reaction mechanisms, and to reason for or against a particular mechanism.201,

202

Thus, the choice of the computational strategy depends on the specific

catalytic step in Pols to be investigated. For example, Markov state models used to investigate major protein conformational changes require extensive sampling (in the s-timescale, and longer),203 which is only accessible by means of force-field based MD simulations. Computationally expensive quantum-based approaches,186, 187 on the other hand, are necessary to examine chemical reactions like, in Pols, proton transfer events and phosphate hydrolysis. That is, each method has its strengths, weaknesses and limitations, usually related to accuracy and sampling. These two variables heavily define the quality of computational investigations of catalytic mechanisms (which often also depends on the researcher’s chemical intuition). Sampling is a major limitation when only a few reaction pathways are investigated. This can be problematic if the sampling omits meaningful regions of the catalytic conformational space, which is not always obvious a priori. Accuracy is also critical, especially when possible pathways return similar energy profiles, making it challenging to compare them. These limitations and challenges are even more difficult when compared to the sophisticated process of enzymemediated nucleic acid processing. With the advent of more powerful computers and algorithms for extended MD simulations and analyses, and advanced approaches to studying catalysis (e.g. machine-learning methods to investigate reaction mechanisms),204 we expect great progress in further elucidating the energetics and dynamics of the complex enzymatic mechanisms of Pol catalysis. Understanding Pol catalysis and mechanism of function is relevant for drug discovery to target native Pols involved in pathophysiological processes. However, it is also relevant in the fast-growing field of artificial Pols design. One such example is the development of new ACS Paragon Plus Environment

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unnatural nucleotides to store and propagate genetic information, with modifications in the sugar ring and in the nucleobases.205 One active area is the design of unnatural nucleotides (referred to as xeno nucleic acids or XNA) to develop synthetic XNA polymers, which can store information and respond to external stimuli, while being immune to endogenous nuclease activity. Another example is XNA polymers that can perform catalysis (xenozymes) for practical applications such as diagnostic and therapeutic technology.206 In this context, computational studies will help increase our understanding of how these artificial objects operate. This will require the development of new force-field parameters and, most likely, new and smart computational approaches to realistically simulate how these synthetic molecules interact with endogenous enzymes. Notably, advances in computational methods and applications will advance the field toward the rational design of artificial Pols, which represent a new frontier in the era of precision genome editing. This Perspective has discussed representative studies that illustrate how computational modelling and simulations have been used to investigate Pol catalysis. These studies have generated insights into the metal-aided mechanisms of enzymatic chemical transformations, which occur in complex multistep reactions catalyzed by Pol enzymes. We conclude by emphasizing that these insights are not only intellectually rewarding but also necessary to interpret experimental data. We expect modern computational methods and approaches to continue to be of help in elucidating the atomic-level motions of Pol action, in enriching the interpretation of existing structural and mechanistic data, and in guiding the development of new experimental studies. For example, computational studies have generated information about the exact mechanism, geometry, and chemico-physical properties of the enzymatic TS of pharmacologically relevant Pols. This information can be used in the design of metal-dependent drugs207 and artificial enzymes that perform biomimetic chemistry, positively impacting human health and technology.208-210 We thus look forward to future computational endeavors that will shed new light on the enzymatic machinery behind the fascinating process of Pol-mediated nucleic acid biosynthesis.


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Acknowledgments M.D.V. thanks the Italian Association for Cancer Research (AIRC) for financial support (IG 18883). V.G. thanks the European Molecular Biology Organization (EMBO) for financial support (ALTF 103-2018). We also thank Grace Fox for proofreading and copyediting the manuscript.


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Figures

Figure 1. Schematic representation of nucleic acid synthesis catalyzed by RNA/DNA polymerases. Precatalytic state, nucleophile activation, nucleotide (d)NTP addition, and nucleic acid translocation with consequent postcatalytic product formation and liberation of pyrophosphate (PPi) leaving group. Green indicates the template strand (T), blue indicates the primer strand (P). The nucleophilic 3’O – is depicted in orange.


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Figure 2. (Left) Overview of the general ternary Pol/(R)DNA/(d)NTP complex. Each Pol domain is differently colored: palm in yellow; thumb in blue; fingers in cyan; little finger in red. (Right) Close view of the two-metal-aided catalytic site commonly found in Pols. The two metal ions are in orange, nitrogen is in blue, carbon is in white, oxygen is in red, and phosphorus is in maroon. The residue K(R), depicted with cyan carbon, identifies the conserved Arg/Lys, which has been systematically found in this conserved position on top of the active site. The K1- and K2-like residues, with green carbon, are basic second-shell residues, which have been discovered in all biomolecules that perform nucleic acid editing.104


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Figure 3. (A) Graph reporting the intramolecular H-bond (indicate as d-PT) in different polymerases. The length of d-PT is reported for structures of Pol families from each domain of life. The X-axis reports the protein name. The Y-axis reports d-PT length (Å). Green dots identify X-ray structures of Pols from prokaryotes, cyan from eukaryotes, and red from viruses. The background color indicates the enzyme commission number (EC number provided above). (B) Superimposition of (ribo)nucleotides co-crystallized in Pol reactive ternary complexes. Structures extracted from different crystals are superimposed following their species (A, C, G, T, U). The upper part indicates the conserved presence of d-PT in those (ribo/deoxy)nucleotides complexed with Pol/(R)DNA binary complexes. The lower part shows the C3′-endo sugar pucker ACS Paragon Plus Environment

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conformation that is always detected in these structures. Ribonucleotides (RNA) are cyan, deoxynucleotides (DNA) are white. The value reported for d-PT is the average value obtained for each type of (ribo/deoxy)nucleotide. Figure adapted from Ref.41.

Figure 4. Reaction mechanisms proposed for nucleotidyl transfer catalyzed by Pols. Horizontal shaded areas highlight the two proposed mechanisms: protein-mediated44 and watermediated and substrate-assisted43 (WMSA). Vertical shaded areas indicate the three major chemical steps for Pol catalysis (i.e. nucleophile activation in the prereactive state, nucleotide addition, and DNA translocation, followed by leaving group departure). Above: the proteinmediated mechanism, whereby a conserved Asp (often part of the widely conserved DED-motif, which is a structural feature of the two-metal-ion mechanism) receives the proton from the nucleophilic group. This proton transfer thus activates the nucleophile for the phosphoryltransfer reaction. The red arrow denotes possible proton transfer events in the proteinmediated mechanism. The dashed green arrow indicates a second plausible nucleophile ACS Paragon Plus Environment

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activation pathway, whereby the proton directly migrates on the non-bridging oxygen of the dNTP -phosphate. Similarly, the dashed cyan arrow depicts an alternative nucleophile activation process mediated by a hydroxide anion in the bulk. Below: the WMSA requires at least two water molecules to mediate the proton shuttle and relay to the -phosphate via the -phosphate and the dNTP as the reaction proceeds.


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Figure 5. Reaction scheme showing the self-activated mechanism (SAM) for nucleic acid polymerization.41 (A) Michaelis–Menten complex: This state leads to the two-metal-aided SN2type phosphoryl transfer with liberation of the pyrophosphate (PPi) leaving group. Notably, the nucleophilic oxygen is already activated here (i.e. deprotonated). (B) Products for nucleotide addition: here, the incoming nucleotide was added to the primer strand. Colored lines indicate selected distances taken as collective variables (CV1 = r1 – r2 and CV2 = r3 – r4 for QM/MM metadynamics) to investigate SAM. (C) Nucleophile formation and nucleic acid translocation: the nucleophile 3′-OH is activated through its deprotonation in favor of the leaving PPi (PT1), while r4 is progressively shortened, indicating initial nucleic acid translocation. (D) PPi exit: at this point, the newly formed 3′-hydroxide group of the incoming nucleotide is coordinated on top of metal A, while the leaving PPi departs from the catalytic site, helped by the transient third ACS Paragon Plus Environment

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metal ion. (E) dNTP binding and catalytic site closure: the enzyme is ready for the subsequent polymerization cycle upon binding of a new nucleotide, with closure of the catalytic cycle. Figure from Ref.41.


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Figure 6. (A, left) Overlap of the structures reported in (B, right) and manually aligned with Coot using the substrate and the two-metal-ion center as a guide.104 Substrate (green cartoon) and MA-MB metals (orange spheres) are reported for endonuclease BamHI only. Blue and red spheres depict the position of the K1- and K2-like elements from all structures, respectively. Grey circles represent spheres of a radius of 4.0 Å (K1) and 3.5 Å (K2) and identify sites 4 (K1) and 3 (K2), respectively. (B) Distances in angstroms of K1-like elements from the acidic residues that coordinate MA-MB (d K1-acidic, blue dots) and of K2-like elements from the substrate (d K2-substrate, red dots). The grey shade covers the optimal range of distances for hydrogen bonds and ionic interactions (Ippolito et al., 1990).211 Several outliers correspond to structures solved at low resolution (i.e. PDB: 5FJ8)212 or with no metals in the active site (i.e. PDB: 1IAW).213 For enzymes in which K1 contacts acidic residues indirectly (PDB: 2BAM,214 1DMU,215 1QPS, and 2ALZ216), we reported the closest distance to the linking residue. For structures where K2 is present,

but

the

substrate

is

not

resolved

in

the

PDB

file

(PDB: 3S1S,217 1FOK,218 4OGC,219 5B2O,220 and 5AXW221), we did not plot any data. In exo-λ structure PDB: 4WUZ,222 the “d K1-acidic” and “d K2-substrate” data points overlap and only the blue dot is actually visible. The names of the enzymes are on the x-axis, corresponding PDB codes are indicated on top of every data point. Enzymes are grouped by classes and their


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respective Enzymatic Classification (E.C.) numbers are indicated on the top of the graph. Figure adapted from Ref.104.


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Figure 7. (A-B) Free-energy surface for SAM in human DNA Pol-η. B, PT1, PT2, C, and D identify saddle points for SAM-catalyzed nucleic acid polymerization in DNA Pol-η, moving from point B of the catalytic cycle to an ensemble of global minima at point D (see reaction scheme and points B and D in Figure 5).41 (C-D) Free-energy simulations of Watson–Crick base pairing (W– C) stability in the presence and absence of K(R) side chain (i.e., Arg61 in Pol-η). Both free-energy surfaces (FESs) were reconstructed using ωPu and ωPy151 as collective variables to energetically describe dATP:dT base-pair stability. In particular, C depicts the FES of wild-type system showing two distinct energetic basins with different ωPu and ωPy values. The deepest corresponds to Watson–Crick base pairing (W–C) while the relative one corresponds to the Hoogsteen base pair (HG). In contrast, D displays the FES of R61A mutant system showing large and deep energetic basins with different ωPu and ωPy values. Here, the deepest energetic minimum


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represents an ensemble of dATP:dT architectures far from the canonical W–C pairing. Of these, HG is the most sampled. In contrast, a relative minimum identifies a cluster of architectures close to the iconic W–C pairing. Figure adapted from Refs.41, 151 Pol Family

Species

Pols

S. cerevisiae Archaeal Archaeal Engineered

DNA Pol-δ KOD DNA-Pol 9°N DNA-Pol KOD-RI TNA Pol

Human

DNA Pol-β

B-family

X-family

DNA Pol-μ Human

DNA Pol-η

Y-family

PDB ID 3IAY113 5OMF111 5OMQ111 5VU8112 [4UAY, 4UB3, 4UB5, 4UBB, 4UBC],5 [4RPY, 4RPZ, 4RQ0, 4RQ2, 4RQ4, 4RQ5, 4RQ6, 4RQ8],109 [4KLG, 4KLH, 4KLI, 4KLJ, 4KLO, 4KLQ],31 [3RH4, 3RH5, 3RH6],125 3JPP223 4M0A,110 [5TYY, 5TYX, 5TYW, 5TYV, 5TYU],33 [5VZ9, 5VZC, 5VZF, 5VZI]224 [4ECT, 4ECU, 4ECV, 4ECW, 4ECX],34 [5KFH, 5KFI, 5KFJ, 5KFK, 5KFL, 5KFN, 5KFP, 5KFW, 5KFX, 5KFZ, 5KG0, 5KG1, 5KG2, 5KG3, 5KG4, 5KG5, 5KG6, 5KG7, 5L9X]47

Table 1. X-ray structures of ternary Pols/DNA/dNTP complexes with three metal ions in their active sites.


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