In crystallo observation of active site dynamics and transient metal ion binding within DNA polymerases

During DNA synthesis, the 3′-OH terminal group of the primer needs to align properly with respect to the Me²⁺ and the incoming nucleotide to undergo deprotonation and nucleophilic attack. Computational simulation studies have revealed that the primer has to be within 2.3–2.8 Å from the α-phosphate for efficient nucleophilic attack (Venkatramani and Radhakrishnan, 2010). A subtle movement or rotation in the primer by only 1–2 Å can place the primer 3′-O beyond this range and restrict chemistry. Moreover, the general base must approach the 3′-OH of the primer to carry out proton transfer before or concurrently during the nucleophilic attack. In order to capture ternary polymerase complexes, most early structural studies utilized DNA in which the primer strand contained dideoxynucleosides at the termini (Doublié , 1998; Sawaya , 1994). Without a functional 3′-OH group, these structures found that the Me²⁺_A was either missing or not properly aligned (Johnson and Beese, 2004; Vaisman , 2005). Thorough examination of primer 3′-OH-Me²⁺_A alignment was first executed with nonhydrolyzable dNTP analogs with X family Pol β (Batra , 2008) and Pol λ (Garcia-Diaz , 2007), revealing that the primer 3′-O coordinates the Me²⁺_A as one of the ligands in the octahedral shell and exists 3.4 Å from the incoming nucleotide prepared for nucleophilic attack. However, exactly how and what docks the primer 3′-O into alignment and promotes its deprotonation and nucleophilic attack remained unclear.

Recent time-resolved x-ray crystallographic studies with X-family Pol β (Freudenthal , 2013; Kumar , 2022; Reed and Suo, 2017; Vyas , 2015), Pol λ (Jamsen , 2022), and μ (Jamsen , 2017), and Y-family Pol η (Nakamura , 2012) uncovered transient events that occur during correct nucleotide incorporation. By complexing Pol η with the correct, natural nucleotide dATP and Ca²⁺, Nakamura and co-workers showed that in the ground state without the Me²⁺_A, the primer lies 4.1 Å away from the α-phosphate group with a bond angle of 169°, slightly misaligned for nucleophilic attack [Fig. 2(a)]. Correlated with the binding of Mg²⁺ at the A-site, the 3′-OH of the primer was docked at 174° by the Mg²⁺_A in the C2′-endo conformation to lie 3.3 Å from the α-phosphate. Coupled with product formation, the deoxyribose sugar ring at the primer end changes from a C2′-endo to C3′-endo sugar pucker conformation (Nakamura , 2012). Similarly, in X-family Pol β and Pol λ, the primer 3′-O shifted over 1 Å closer to the α-phosphate upon Ca²⁺/Na¹⁺ to Mg²⁺/Mn²⁺ exchange at the Me²⁺_A binding site (Freudenthal , 2013; Jamsen , 2022; Kumar , 2022). These results highlight the contribution of Me²⁺_A binding and its role on primer 3′-O alignment.

What exactly promotes primer deprotonation and whether it concertedly occurs with nucleophilic attack remain unclear and critical in understanding the catalytic mechanism of polymerases. During the in crystallo reaction with X-family Pol λ, a water molecule lying close to the primer terminus was captured, suggesting its potential role to aid primer deprotonation (Jamsen , 2022). In contrast, in crystallo reaction by Pol β did not capture a water molecule interacting with the 3′-OH of the primer (Freudenthal , 2013). Instead, it was proposed that the amino acid side chain Asp256, which lies close to the primer, might be responsible for primer deprotonation. Computational studies indicate that Asp256 might facilitate primer deprotonation (Batra , 2013). However, it is important to note that this amino acid residue also coordinates the Me²⁺_A, and mutating Asp256 will disrupt Me²⁺_A binding and also indirectly affect deprotonation. In the study with Y-family Pol η, occurring simultaneously with Me²⁺_A binding, a water molecule approached from behind and around the 2′ carbon of the primer to possibly facilitate 3′-OH deprotonation (Nakamura , 2012). The protein residue Ser113, which lies close to the primer but does not coordinate the Me²⁺_A, has also been suggested to promote primer deprotonation. In such close proximity to the primer terminus, the water molecule or Ser113 can easily accept the primer 3′-O proton. However, simultaneously displacing the water molecule and mutating amino acid residue 113 from serine to an alanine did not reduce catalytic efficiency (Gregory , 2021). Therefore, primer deprotonation in Pol η was proposed to most likely occur through multiple pathways, and a well-defined general base is not needed.

During the incorporation of the incorrect nucleotide, the role of the Me²⁺_A and the importance of primer terminus alignment were further pronounced. In static crystal structures of Pol η with the incorrect nucleotide at the insertion site, the primer is positioned misaligned in the up-shifted conformation, restricted from attacking the α-phosphate (Zhao , 2013). A water molecule coordinates the Me²⁺_A in place of the primer 3′-O. Notably, the degree of primer misalignment varies depending on the DNA sequence near the primer terminus, which corresponds to the different misincorporation efficiencies reflected in the kinetic assays (Zhao , 2013). During the in crystallo reaction with Pol η, the majority of the primer terminus exists in the misaligned up-shifted conformation near the roof of the active site in the absence of Me²⁺_A (Chang , 2022). Upon Mg²⁺ binding to the Me²⁺_A binding site, the primer terminus is docked in the aligned conformation [Fig. 2(b)]. Nevertheless, the primer terminus exists 2.5 Å away from the Me²⁺_A, still 0.5 Å longer than the optimal distance for Mg²⁺ coordination, suggesting major barriers in primer deprotonation and alignment. Similarly, during the in crystallo reaction with Pol β incorporating a dATP across a dG template, the primer terminus showed weak electron densities (σ less than 3.0 r.m.s.d for the F_o-F_c omit map), suggesting it is partially destabilized (Freudenthal , 2013). The suboptimal coordination distance of the Me²⁺_A to the primer end and this misaligned conformation in both polymerase systems, which places the primer terminus 3′-O in an unfavorable position for nucleophilic attack, explain the significant decrease in catalytic activity during misincorporation. Furthermore, it was previously established that Mn²⁺ can promote error-prone synthesis of polymerases. Titrating the polymerases in crystallo revealed that Mn²⁺ was more optimal than Mg²⁺ at aligning the primer end in Pol η, and binding of Mn²⁺ linearly correlates with primer alignment even during incorrect nucleotide incorporation (Chang , 2022). Such mutagenic property of Mn²⁺ was also previously hinted by static crystal structures of Pol λ. The structures of Pol λ incorporating dCTP against a dG revealed that Mn²⁺ promotes the sugar pucker to exist in the C3′-endo conformation as opposed to the C2′-endo conformation, aiding the primer 3′-O to exist even closer to the target α-phosphate (Garcia-Diaz , 2007). These results further highlight a correlation between the Me²⁺_A, primer alignment, and polymerase fidelity.

Rearrangements at the primer terminus and the Me²⁺_A coordination environment in the polymerase active site have been observed during DNA damage bypass and nucleotide analog drug insertion. Phenanthriplatin is a potential cisplatin-analog cancer drug that covalently links to DNA and obstructs DNA synthesis and transcription (Johnstone , 2014; Kellinger , 2013; Park , 2012). Similarly, cyclopurines like 8,5′-cyclo-2′-deoxyadenosine also stall the DNA replication machinery (Kuraoka , 2001). When Pol η contacts 8,5′-cyclo-2′-deoxyadenosine or phenanthriplatin on the DNA template, the primer was found to misalign, preventing nucleotide incorporation (Gregory , 2014; Weng , 2018). The primer end aligned with the incoming nucleotide, and bypass occurred only in the presence of mutagenic Mn²⁺. Similarly, the drug (-)-β-L-2′,3′-dideoxy-3′-thiacytidine (lamivudine) binding at the Pol β insertion site resulted in primer terminus misalignment and perturbation of the Me²⁺_A octahedral coordination geometry. These structural results were reflected in lamivudine's tighter binding affinity and 323-fold less efficient incorporation rate than the natural substrate dCTP (Vyas , 2017). In Pol β, Arg283 stabilizes the syn-conformation of 8-oxo-2′-deoxyguanosine (8-oxodG) through hydrogen bonding on the template. Such interaction shifts 8-oxodG into a geometry that favors base-pair binding of dATP, allowing dCTP to be incorporated over dATP by only 2-folds. When Arg283 is mutated to Lys in Pol β, 8-oxodG existed in the anti-conformation and shifted the template upstream by 2 Å, tugging the primer away from the Me²⁺_A to exist 139° out of line with the α-phosphate unable to attack the incorrect nucleotide dATP (Freudenthal , 2012). Thus, primer alignment might be an innate mechanism that safeguards against the bypass or incorporation of some damaged substrates.

Furthermore, chemical modifications on the sugar moiety of nucleotides can affect the dynamics of primer alignment and incorporation efficiency. When a ribonucleotide (rN), which has one additional oxygen atom on C2′ compared to a deoxynucleotide (dN), existed at the primer terminus, the primer terminus was found to be in the aligned conformation even in the absence of the Me²⁺_A during correct nucleotide incorporation of Pol η (Gregory , 2021). Recent studies show that misincorporation is favored during Pol η-mediated extension from a rN (Chang , 2023). Crystal structures with rN at the 3′-end of the DNA duplex showed improvement in primer alignment even when the incorrect dGTP nucleotide was bound at the insertion site across from a template dT. Not only was the primer terminus more favored in the docked position for nucleophilic attack, but also the sugar motifs of these rN existed in the C3′-endo sugar pucker conformation, which is the favored conformation in the product state, as the 3′-O of the primer lies closer to the target α-phosphate (Batra , 2006; Berman , 2007; Doublié , 1998; Franklin , 2001; Garcia-Diaz , 2007; Li , 1998; Nakamura , 2012; Wang and Yang, 2009). Cytarabine (araC) is a dC mimic that contains a -OH group in the β direction on the 2′ carbon of the arabinose sugar moiety. Static structures show that at the primer terminus, araC exists in both aligned and misaligned conformations even during correct nucleotide incorporation. Moreover, this dC mimic drug exists in the C2′-endo conformation and misaligned with the α-phosphate, explaining how araC inhibits DNA extension even though it contains a functional 3′-OH group (Rechkoblit , 2018). These results again showcase the importance of primer alignment and suggest small changes on the sugar ring can significantly impact incorporation efficiency.

Based on static crystal structures, Steitz proposed that two-metal ions are required and sufficient for polymerase catalysis (Steitz and Steitz, 1993). The Me²⁺_B stabilizes the incoming nucleotide by coordinating with its phosphate oxygens; the Me²⁺_A helps to decrease the pKa of the primer for deprotonation and nucleophilic attack. Recent in crystallo reaction studies revealed an additional divalent metal ion, or the C site metal ion (Me²⁺_C) binds between the Pα and Pβ of the incoming nucleotide during reaction (Freudenthal , 2013; Nakamura , 2012; Vyas , 2015; Yang , 2016). Instead of protein sidechains, it is coordinated by four water molecules and two oxygens on the newly formed DNA strand and pyrophosphate. It has only been captured during catalysis but not detected in static structures with various inhibitors. Thus, the Me²⁺_C was not detected in decades of static structures of polymerases and remains hard to probe. The exact timing of Me²⁺_C binding and its role in DNA synthesis remain controversial. It has been proposed that the Me²⁺_C stabilizes the product state, pushes the reaction forward, inhibits the forward reaction, induces the reverse reaction, and/or helps with damage bypass.

Initially, the Me²⁺_C has been suggested to bind to the newly formed DNA and pyrophosphate to stabilize the product state. In Pol η, the Me²⁺_C was captured simultaneously with production formation, except at the very beginning of the reaction (Nakamura , 2012). In Pol β (Freudenthal , 2013) and μ (Jamsen , 2017), during correct nucleotide incorporation, the electron density of the Me²⁺_C appears slightly right after the detection of new bond density. Concurrently or immediately after, the Me²⁺_A dissociates from the active site. Thus, the role of the Me²⁺_C has been suggested to help reduce the electronegative buildup of the newly formed DNA backbone and also to help induce pyrophosphate protonation and release [Fig. 3(b)].

Moreover, the Me²⁺_C potentially drives the reverse reaction of phosphorolysis. The Me²⁺_C coordinates the newly formed DNA and pyrophosphate oxygens at 2.0–2.2 Å. Just like how the Me²⁺_A closely coordinates the primer terminus and helps to deprotonate the primer 3′-OH, the Me²⁺_C is optimally positioned to deprotonate one of pyrophosphate's oxygen. Once deprotonated, the more electronegative charged oxygen can undergo nucleophilic attack on the newly formed product phosphate to break the newly formed bond. Hence, it has been suggested that the role of the Me²⁺_C lies in deprotonating pyrophosphate (Freudenthal , 2013). Nevertheless, phosphorolysis has never been visualized with time-resolved crystallography before, as the forward reaction is far more efficient than the reverse reaction. However, quantum mechanical/molecular mechanical simulations show that the Me²⁺_C can reduce the energy barrier for phosphorolysis to occur (Perera , 2015).

Recent evidence suggests that the Me²⁺_C may be required to drive the DNA synthesis reaction. With Mn²⁺, which is more electron dense and, thus, can be detected at low occupancies, it was shown that Me²⁺_C binding perfectly correlates with production formation even during early stages of the reaction in Pol η (Gao and Yang, 2016). Recent studies with cross-linked Pol β also showed the Me²⁺_C binding simultaneously with phosphodiester formation (Kumar , 2022). Moreover, extensive in crystallo titration with Pol η found that the A and B sites are high affinity binding sites (<0.5 mM), while the C site is a weak binding site with a low binding affinity of ∼3 mM, similar to what has been found in solution. When only the A site and B sites are saturated, the primer 3′-O, α-phosphate, and bridging oxygen co-linearize and are inclined for nucleophilic attack, but the reaction product is not observed even after long reaction time. Only after Me²⁺_C binding, product formation did occur, suggesting the essential role of the Me²⁺_C during catalysis. Furthermore, when a sulfur element, which is less electronegative than oxygen, replaces the incoming nucleotide's Sp oxygen, which serves as a coordinating ligand to the Me²⁺_C, density surrounding the usual Me²⁺_C is not captured, and the Me²⁺ binding affinity is reduced by 25-fold from the kinetic assays (Gao and Yang, 2016). During slow mismatch incorporation, the transient Me²⁺_C was captured binding before product formation (Chang , 2022). Initially, it binds between the Pα and Pβ oxygens but exists slightly too far from the Pα oxygen of the incoming nucleotide. After the Me²⁺_C enters the active site in between the Pα and Pβ oxygens and shifts downward to form an optimal octahedral geometry, the α-β-phosphate bond breaks. Thus, the in crystallo studies with Pol η suggested that Me²⁺_C binding proceeds catalysis, and that the Me²⁺_C may be directly involved in reducing the energy barrier for α-β-phosphate bond breakage [Fig. 3(b)].

This proposed catalytic role of the Me²⁺_C has been challenged in recent years. Wang and colleagues point out that because the occupancy of the Me²⁺_C is always lower than that of the product pyrophosphate, the Me²⁺_C must bind after reaction in the product complex and, thus, cannot actively push the catalytic reaction forward (Wang and Smithline, 2019). However, it has been argued that time-resolved experiments performed by hand cannot differentiate whether the Me²⁺_C binds before or after reaction, as Me²⁺_C binding can occur on the order of sub-seconds (Tsai, 2019). They also argued that the loss of the Me²⁺_C binding after product DNA and pyrophosphate formation could explain why the occupancy of the Me²⁺_C is always lower than that of the product pyrophosphate. While computational studies argue that Mg²⁺ or Mn²⁺ cannot favorably bind between the Pα and Pβ oxygens of the incoming nucleotide due to suboptimal bond lengths, geometry angle, and electrostatic charges of the oxygens, Chang and colleagues captured Mg²⁺ and Mn²⁺ binding near the Me²⁺_C binding site prior to reaction (Chang , 2022). Furthermore, high concentrations of Mg²⁺ or Mn²⁺ used in in crystallo soaking experiments have been shown to inhibit the polymerase reaction in solution (Frank and Woodgate, 2007; Wang and Konigsberg, 2022). Thus, the Me²⁺_C has been suggested to remain bound with the product pyrophosphate within the active site. At this location, it can induce the reverse reaction and hold the product pyrophosphate within the active site. With the substrate unable to enter, additional DNA synthesis cannot occur at high metal ion concentrations.

It has also been proposed that transient divalent metals can help during the incorporation of damaged substrates. During the incorporation of the anti-form of 8-oxo-7,8-dihydro-2′-deoxyguanosine triphosphate (8-oxoGTP), which is sterically destabilized within the nucleotide binding pocket in Pol β, minimal distortion of the active site was observed. Instead, a divalent metal ion was captured binding near the Pα and Pβ oxygens prior to reaction to possibly alter the electrostatic nature of the α-phosphate oxygens to favor nucleophilic attack (Freudenthal , 2015). However, another study on dCTP incorporation across an (anti) 8-oxoGTP found the Me²⁺_C only binds during the catalytic reaction (Vyas , 2015). Similarly in X-family Pol μ, after crystals were soaked in 50 mM Mn²⁺, density close to the Me²⁺_C was captured and found coordinating the C8′ oxygen of 8-oxodGTP and a nearby aspartate indirectly through a water molecule (Jamsen , 2021). Furthermore, an additional divalent metal site was captured near the Me²⁺_A binding site in Pol η during the incorporation of Sp-dATPαS, which has the Sp oxygen replaced with a sulfur element (Gao and Yang, 2016). While it remains unclear if these densities found in Pol β, μ, and η truly represent the Me²⁺_C, it is likely that one additional or more transiently bound divalent metal ions play the role of stabilizing strained substrates for more efficient incorporation.

In addition to DNA polymerases, it has also been proposed that all RNA polymerases (Steitz, 1998), many nucleases (Stahley and Strobel, 2005; Toor , 2009; Toor , 2008), and ribozymes (Steitz and Steitz, 1993) follow the two-metal-ion dependent catalysis mechanism. Similar time-resolved crystallography experiments revealed an additional divalent Me²⁺ binding transiently during RNA cleavage in RNaseH (Samara and Yang, 2018). Moreover, the authors further discovered two additional monovalent Me¹⁺ binding near the RNA backbone, possibly facilitating the water-mediated deprotonation and nucleophilic attack as well as stabilizing the transition state. The existence of the third Me²⁺ has also been suggested by studies on Endonuclease V, which was proposed to use a two-metal-ion dependent mechanism for cutting RNA. Similar as in RNaseH, a third divalent Me²⁺ binds transiently to direct the nucleophilic water molecule during Endonuclease V catalysis (Wu , 2019). Furthermore, a transient Me²⁺ has also been sighted in MutT, which initially was proposed to catalyze 8-oxodGTP hydrolysis with two divalent Me²⁺ (Nakamura and Yamagata, 2022). The intermediate structures showed an additional metal ion binding in between two protein residues and directing a nucleophilic water closer and aligned to the scissile phosphate. In each of these three systems, the transient divalent Me²⁺ binds on the side of the nucleophilic water molecule, aligning it for nucleophilic attack. It is possible that many more metalloenzymes use transient metal ions during catalysis to stabilize substrate, product, and/or promote transition state formation.

Time-resolved crystallography has extensively increased the understanding of DNA polymerases by revealing transient events like primer alignment, role of the Me²⁺_A, and the presence of transient metal ions that occur during nucleotidyl transfer and DNA repair. However, in crystallo visualization of DNA synthesis remains limited to only X- and Y-family polymerases, which are specialized in DNA repair and damage bypass, possibly because polymerases that function during DNA replication such as A- and B-family polymerases perform catalysis too rapidly to be observed in crystallo. Furthermore, polymerases that exhibit large global conformation changes, diffract to sub-atomic resolutions lower than 3 Å, or are intolerant of cofactor soaking would present challenges for in crystallo studies. In addition, this manual metal ion diffusion technique is limited by how fast the crystallographer can feasibly transfer ternary complex crystals between buffer solutions, which is typically limited to 10 s. Automated stop flow equipment coupled with x-ray free electron lasers may further improve the temporal resolution of transient observations and lead to discoveries of new protein dynamic events before or during catalysis (Olmos , 2018). Nevertheless, the number of diverse enzymes being studied with time-resolved crystallography continues to increase, highlighting the essential roles of active site dynamics and transient element binding during enzyme catalysis.

This work was supported by CPRIT (No. RR190046) and the Welch Foundation (No. C-2033–20200401) to Y.G. and a predoctoral fellowship from the Houston Area Molecular Biophysics Program (NIH Grant No. T32 GM008280, Program Director Dr. Theodore Wensel) to C.C.

The authors have no conflicts to disclose.

Caleb Chang: Writing – original draft (equal); Writing – review & editing (equal). Grace Zhou: Writing – review & editing (equal). Yang Gao: Writing – original draft (equal); Writing – review & editing (equal).

The data that support the findings of this study are available from the corresponding author upon reasonable request.