DNA Repair and Fidelity Mechanism

 

The genome, contained within the DNA in the nucleus of cells, is the master blueprint for an organism.  Therefore, it is a startling fact of life that DNA in every cell of the human body is spontaneously damaged more than 10,000 times every day [Lindahl, 1993].  To circumvent this problem, cells have evolved sophisticated machinery to replicate and repair DNA accurately and efficiently.

 

DNA polymerases orchestrate the addition of new nucleotides to a growing chain of DNA by catalyzing the nucleotidyl-transfer reaction which increases the primer strand by one base at a time.  The relative ability of a polymerase to incorporate a correct nucleotide rather than an incorrect unit from a pool of structurally similar molecules is a measure of fidelity. When an incorrect nucleotide (e.g., G opposite A instead of T) is inserted, the blueprint directing the cell is changed. Since the accumulation of such errors is thought to play a key role in the aging process and many disease states [Beard & Wilson, 1998], understanding polymerase mechanisms at the atomic level is very important.

 

DNA polymerases are found from primitive viruses and bacteria to complex multi-cellular organisms, including human beings. Various families of DNA polymerases have evolved, and many play different roles in the same organism. For instance, research has found that humans possess multiple DNA polymerases.  Based on sequence and structural similarities, seven different DNA polymerase families have been identified. Because they are crucial for maintaining genomic integrity, these polymerase families are evolutionarily conserved and members are often found in a wide range of organisms. 

 

Our work focuses on DNA repair polymerases from the X and Y-family that lack intrinsic proofreading abilities.  Unlike the high fidelity DNA replication polymerases, these lower fidelity enzymes typically have specialized functions and only insert a few bases at a time.  Some of these polymerases are involved in the repair of oxidative lesions and double strand breaks while others like Dpo4 specialize in lesion bypass.  High fidelity polymerases often stall when they encounter a bulky DNA lesion, but bypass polymerases can rescue replication and insert bases opposite to the lesion or extend lesioned base pairs.

 

In our work, we study a representative group of repair DNA polymerases that includes X-family DNA polymerases β (pol β), X (pol X), and λ (pol λ) ; and Y-family DNA polymerase IV (Dpo4).  Each polymerase has a characteristic fidelity and error specificity.  Our research focuses on their structure-function relationships and the various mechanisms that ensure replication fidelity. Although many high resolution X-ray crystal structures are available for these and other DNA polymerases, they only provide a static snapshot of the enzyme while interpreting how polymerases work often requires a dynamic picture. Experimental kinetic data are available, with information on the behavior of polymerases on the macroscopic level (such as fidelity and efficiency rates, error characteristics, etc.), but often insights are required at the atomic level.

 

Computer simulations can help bridge the gap between X-ray structures and observed characteristics by providing critical information on how polymerases operate at the atomic level. While subject to certain well-known limitations such as imperfect force fields and limited sampling, molecular dynamics (MD) simulations based on the structures can help reveal the motions of these enzymes upon substrate binding or removal.  However, molecular dynamics simulations based on classical force fields alone (i.e., molecular mechanics, MM) are not sufficient to study the nucleotidyl transfer reaction because it involves covalent bonds breaking and forming. Thus, the chemical reaction is simulated for highest accuracy using quantum mechanics and the remaining environment modeled using classical MM. This mixed QM/MM representation has proven quite useful in studying both details of the catalytic reaction and the effect of the remaining protein, DNA, and solvent environment.

 

Computational Studies on DNA Polymerase β

 

DNA polymerase β (pol β) fills short DNA gaps during repair synthesis in the base excision repair (BER) pathway and plays a role in chromosomal replication [Beard & Wilson, 1998]. DNA pol β is composed of two domains: lyase (8-kDa) and polymerase (31-kDa). The shape of the latter is like a hand with fingers, palm, and thumb subdomains [Ollis et al., 1985]. This is a common architecture for DNA polymerases [Pelletier et al., 1994; Li et al., 1998; Kiefer et al., 1998; Ding et al., 1998; Huang et al., 1998; Doublie & Ellenberger,1998; Sawaya et al., 1997]. Structural and kinetic evidence suggests that pol β selects the correct deoxynucleoside triphosphate (dNTP) from a pool of structurally similar molecules through an ‘induced fit’ mechanism by alternating motions between open and closed states: substrate binding and product release occur in the open state, while the chemical DNA extension occurs in the closed state [Beard & Wilson, 1998].

 

Our group has worked in collaboration with leading experts in the chemistry and biology of DNA polymerases to elucidate the mechanisms used by these enzymes for replication and repair of normal and damaged DNA. In particular, our group has established novel protocols to investigate fidelity mechanisms of mammalian pol β.  These studies have revealed detailed aspects of the conformational and chemical events that provide the enzyme with a series of checks and balances, which allow it to discriminate between correct and incorrect base pairs and hence control fidelity.

 

Slow local rearrangements in pol β's conformational pathway. Details of the precise events involved in pol β’s large-scale opening/closing motions and how they may regulate synthesis fidelity have been investigated experimentally. In particular, kinetic data reveal slow conformational steps before and after chemistry (steps 2 and 4 of the catalytic cycle in Fig. 1).  Enzyme kinetic measurements, however, suggest that the subdomain opening/closing motions themselves are relatively fast [Vande Berg et al., 2001]. 

 

We set out to identify the nature of the slow molecular rearrangements that prepare the active site for the chemical step.  By a combination of modeling and methodology advances, we simulated the partial opening of pol β after chemistry [Yang et al., 2002a] started from an intermediate structure constructed as average of the crystallographic coordinates of the ternary closed and binary open pol β forms. Together with complete opening by high-temperature and targeted MD simulations from a closed structure, we proposed a sequence of events during pol β’s opening after chemistry: (1) Phe272 ring flips away from Asp192, (2) large thumb movement, and (3) Arg258 rotates toward Asp192. Our estimated rate constant for the Arg258 rotation (~2 × 10^-2 s^-1) suggests that the Arg258 rotation is slow relative to subdomain motions (i.e., thumb opening/closing). Subsequent MD [Arora et al., 2005] and transition path sampling [Radhakrishnan & Schlick, 2004a] simulations for the subdomain closing motions also corroborate that a large free energy barrier is associated with Arg258’s rotation. The local Arg258 motion that we identified may be a crucial component in the orchestration of the active-site in the overall kinetic pathway and therefore may steer the system to the correct state required for chemistry.  While chemistry may be rate limiting overall, the conformational change directs the enzyme to the crucial active site geometry.

Figure 1. Schematic drawing of pol β's synthesis cycle.

 

Reaction pathways for pol β’s conformational change delineated using transition path sampling and evidence for induced fit.  Our studies of the conformational change pathway of pol β provided evidence for the induced-fit mechanism [Arora et al., 2004; Arora et al., 2005a], in which the correct incoming base triggers the requisite conformational change while an incorrect incoming nucleotide hampers the process (Fig. 2).  We also studied dNTP binding specificity and polymerase fidelity by simulating the closing conformational transition of pol β for G:G, G:T, and T:T using standard MD simulations [Arora et al., 2005b] and G:C and G:A nascent base pairs [Radhakrishnan & Schlick, 2005] using transition path sampling. A key feature of our implementation was the enumeration of multiple transition regions by a novel divide and conquer approach [Radhakrishnan & Schlick, 2004a] and an efficient new free energy method we termed “BOLAS” [Radhakrishnan & Schlick, 2004b].

 

Figure 2. C_a traces of superimposed pol β/DNA complex with dCTP (top left) and without dCTP (bottom left) for the intermediate starting structure (yellow), crystal closed (red), and crystal open (green) and the trajectory final structures (blue) [Arora & Schlick, 2004]. Notable are the residue motions in the thumb subdomain and the 8-kDa domain. The positions of a-helix N in the simulated systems are compared to the crystal structures in panels on the right (top, with dCTP, and bottom, without dCTP)

 

The complex reaction profile as delineated in [Radhakrishnan & Schlick, 2004a] reveals five transition-state regions –– partial thumb closing, Asp192 flip, Arg258 rotation, Phe272 flip, and rearrangement of catalytic region –– and relative free-energies involved (Fig. 3).  Significantly, the free energy barrier for the rate-limiting conformational step, namely Arg258 rotation (19±3 k_BT), suggests that conformational steps direct the system to the reaction-competent state in a substrate-sensitive manner.  Studies on the G:A (Fig. 4) mismatch suggest that the closed state (in contrast to the  G:C case) is unstable, and that multiple pathways are involved in the overall pathway. Moreover, the differences in the evolution of key distances between the nucleophilic O3' anion (the reactive group of the DNA primer) with catalytic Mg²⁺ and O3' – P_α of dNTP, as well as interactions associated with Arg283, may affect fidelity mechanisms.

 

Figure 3.  Left: Molecular snapshots near open (left column) and closed (right column) states of pol β for four transition state regions [Radhakrishnan & Schlick, 2004a]: (1) Partial thumb closing. (2) Asp-192 flip. (3) Arg-258 partial rotation. (4) Phe-272 flip. Right:  Overall captured reaction kinetics profile (from TPS) for the conformational transition of pol β (for G:C) from open (state 1) to closed (state 7) forms showing free energies (in k_BT) associated with the different transition state regions [Radhakrishnan & Schlick, 2004a]. The metastable basins (in red) along the reaction coordinate are numbered 1-7. See animated sequence of this closing on our website: http://www.biomath.nyu.edu/index/gallery.html.

 

 

Figure 4. Overall captured reaction kinetics profile for pol β's closing transition followed by chemical incorporation of dNTP for G:C and G:A systems [Radhakrishnan & Schlick, 2005]. The barriers to chemistry (dashed peaks) are derived from experimentally measured k_pol values.  The profiles were constructed by employing reaction coordinate characterizing order parameters in conjunction with transition path sampling. The potential of mean force along each reaction coordinate is computed for each conformational event.

 

Our study of the roles of the two Mg²⁺ ions on both pol β’s conformational closing and opening pathways suggested that closing before chemistry requires both divalent metal ions in the active site while opening after chemistry is triggered by release of the catalytic metal ion  [Yang et al., 2004b]. The closed conformation is stabilized by the interaction of the incoming nucleotide with conserved catalytic residues (Asp190, Asp192, Asp256) and the two functional magnesium ions. These subtle and slow geometric adjustments of both magnesium ions help to guide polymerase selection for the correct nucleotide.  Since Mg²⁺ and Na⁺ ions can diffuse into the active site relatively rapidly, we suggest that the binding of the catalytic Mg²⁺ itself is not a rate-limiting conformational or overall step.

 

Effects of DNA mispairs and key protein residues on pol β’s opening and active-site geometries. Our analyses of pol β/DNA complexes with G:G, C:C, and A:C (template-primer) nascent mispairs after chemistry from dynamics simulations suggested subtle differences and contributions of local distortions at the active site and local conformational rearrangements to polymerase fidelity  [Yang et al., 2002b].  These mismatch studies revealed distorted geometries in the active site with a hierarchy (G:G > C:C > A:C), paralleling the experimentally-deduced inability of pol β to extend these mismatched base pairs and in agreement with geometric selection models proposed by Echols and Goodman  [Echols & Goodman, 1991]. This suggests that the greater distortions in the active site likely impede the DNA extension process. Since pol β does not have intrinsic proofreading activity, such local distortions of the binding pocket could slow DNA extension and lead to external proofreading by an exonuclease. Beyond these deformations, our transition path sampling applications to the closing of pol β before chemistry suggest very different conformational landscapes for G:C versus G:A mismatch systems (Fig. 4).

 

Key protein residues near the active site affect polymerase fidelity, catalytic efficiency, and nucleotide binding efficiency in DNA synthesis [Kunkel & Bebenek, 2000]. We investigated the conformational transition behavior of five single mutants: Arg283Ala, Tyr271Ala, Asp276Val, Arg258Lys, and Arg258Ala by dynamics simulations [Yang et al., 2004a]. Analyses unravel structural adjustments due to single-residue mutation.

 

Pol β’s chemical mechanism for G:C vs. G:A systems. In addition to exploring the conformational pathways, we have studied pol β's mechanism for catalyzing the nucleotidyl-transfer reaction of correct and incorrect insertions using both quantum mechanics (QM) and combined quantum mechanics and molecular mechanics (QM/MM) approaches [Radhakrishnan & Schlick, 2006b; Alberts, et al., 2007; Bojin & Schlick, 2007]. The proposed pathways for the G:C and G:A systems support a series of transient intermediates involving a Grotthuss hopping mechanism of proton transfer between water molecules and the three conserved aspartate residues in the enzyme’s active site [Radhakrishnan & Schlick, 2006b] (Fig. 5).  In the G:C system, the rate-limiting step is the initial proton hop with a free energy of activation of at least 17 kcal/mol, which corresponds closely to measured k_pol values. Fidelity discrimination in pol β can be explained by a significant loss of stability of the closed ternary complex of the enzyme in the G:A system and a much higher activation energy of the initial step of nucleophilic attack, namely deprotonation of the terminal DNA primer O3'H group. Thus, subtle differences in the enzyme active site between matched and mismatched base pairs generate significant differences in catalytic performances. Our related studies also emphasize the variability in pathways for the chemical reaction, depending on the initial state and the environment (ions, solvations).

Figure 5. Left: Schematic drawing of the mechanism of concerted proton-hops during phosphoryl transfer in pol β [Radhakrishnan & Schlick, 2006b]. Right: Captured reaction intermediates for pol β's phosphoryl transfer in the G:C system [Radhakrishnan & Schlick, 2006b]. Key distances A = O3' – P_a and B = P – O3A are provided: (a) reactant state, A = 2.90 Å, B = 1.7 Å; (b) first intermediate, A = 1.94 Å, B = 1.97 Å; (c) second intermediate, A = 1.73 Å, B = 2.0 Å; (d) third intermediate, A = 1.73 Å, B = 2.41 Å; (e) fourth intermediate, A = 1.70 Å, B = 5.53 Å; (f) product, A = 1.70 Å, B = 5.8 Å. The colors represent: cyan (D256), red (D190), blue (D192), pink (dCTP), green (CYT: terminal DNA primer), black (the O3'H-proton), yellow (the O3' oxygen, attacking nucleophile), tan (central phosphorous), purple (leaving O3A oxygen), and orange (Magnesium). The oxygens and hydrogens of water molecules are in red and white, respectively. The arrows denote the location and direction of proton hop.

 

Evidence of Induced-fit Mechanism in African Swine Fever Virus Pol X

 

We also compared DNA polymerase fidelity mechanisms between a polymerase like pol β found in higher mammals with a polymerase from a virus by simulating African Swine Fever Virus DNA pol X [Sampoli Benitez et al., 2006].  This enzyme is a member of the X-family that may participate in BER.  Unlike other X-family enzymes that we have studied, pol X lacks the fingers subdomain.  Interestingly, two NMR structures of free pol X are available that show the enzyme “ open ” and “ close &rdquo conformations [Showalter et al., 2001; Maciejewski et al., 2001].  To examine the effect of salt concentration on the protein conformation, we simulated both structures and found that they interconvert upon salt concentration changes: at physiological low salt conditions the open form is favored while at high salt the more closed form is preferred.  To investigate pol X’s interactions with its substrates, we modeled pol X/DNA and pol X/DNA/dNTP complexes.  Simulations of these complexes revealed that pol X/DNA complexes favor an open conformation distinct from the open conformation assumed by free pol X and pol X/DNA/dNTP complexes favor a more closed conformation, indicating that pol X follows the induced-fit mechanism upon correct substrate binding. Moreover, the closing motion of the thumb is accompanied by several active-site residue rearrangements that prepare the active site for the chemical reaction. Thus, similarities exist between the catalytic cycles of a viral enzyme like pol X and a mammalian enzyme such as pol β.

 

Complex Connections between Structure, Flexibility, and Fidelity in Pol λ

 

Pol λ is a low to moderate fidelity member of the X-family of DNA polymerases.  A comparative analysis of this polymerase with another X-family enzyme, pol β, provides an opportunity to link structural differences with variations in observed fidelity characteristics.  Although pol λ and pol β share many similarities including a striking structural homology, the two enzymes have several divergent properties.  Experimental evidence suggests that pol λ participates in the repair of double strand breaks through the nonhomologous endjoining pathway and also may play a back up role for pol β in BER [Moon et al., 2007]. 

 

Pol λ’s conformational pathway. In agreement with recent X-ray crystal data [Garcia-Diaz et al., 2005], our MD simulations suggest that pol λ does not demonstrate large-scale subdomain movements like pol β [Foley et al., 2006].  However, significant DNA motion exists, and there are sequential side-chain motions associated with Arg514, Arg517, Ile492, Phe506, and Tyr505, all coupled to active site divalent ions and DNA motion (Fig. 6).  Collectively, these motions transform pol λ to the chemistry-competent state.  As proposed for pol β, motions of these residues may serve as gate-keepers by controlling the evolution of the reaction pathway before the chemical reaction.

 

Pol λ’'s slippage tendency. These intriguing results and their implications regarding fidelity stimulated us to investigate the atomic-level basis of pol λ's unusual error specificity:  the generation of far more single base deletion errors than base substitution errors [Bebenek et al., 2003].  Pol λ’s propensity for deletions even exceeds the error rates of Y-family enzymes [Bebenek et al., 2003; Garcia-Diaz & Kunkel, 2006]. X-ray crystal data on pol λ suggests that these deletions occur through DNA template strand slippage [Garcia-Diaz et al., 2006]. To elucidate critical interactions that constrain the DNA in the active ternary position, we simulated mutant derivatives of pol λ at position 517 and compared them to wild-type trajectories [Bebenek et al., 2008; Foley & Schlick, 2008].  Our results indicate that discrete orientations of the mutant residues can occur and that these orientations impact protein-coupled DNA stability by forming unfavorable electrostatic interactions; the less stable these interactions, the lower the stability of the ternary complex and the more movement toward the binary conformation.  The extent of DNA movement in our simulations is mutant dependent and mirrors the deletion error rates already determined for wild-type pol λ and the Lys and Ala mutants [Bebenek et al., 2008].  Besides demonstrating Arg517's important role in driving the catalytic reaction of pol λ, our studies help in understanding pol λ’s slippage tendency by showing the critical role played by residue 517’s interactions with the DNA. Our studies demonstrate that pol λ’s unique architecture may facilitate deletion errors because of the tenuous nature of these specific protein/DNA interactions compared to other DNA polymerases.

 

Pol λ’s error characteristics and chemical mechanism. Our future research is designed to further explore pol λ’s fidelity mechanisms. We will delineate pol λ’s conformational insertion pathways for the correct incoming nucleotide with and without a slipped DNA template strand (e.g., with extrahelical nucleotide) as well as for the incorrect incoming nucleotide without any DNA misalignment.  This work will elucidate the structure/interaction/flexibility of pol λ’s active site and explain pol λ’s accommodation to these various substrate contexts.  We will also describe the chemical reaction catalyzed by pol λ in the different substrate contexts.  We will utilize QM/MM calculations to provide a detailed description of the energetic barriers and steps involved in the reaction pathway and elucidate how chemistry can occur when the template is misaligned or an incorrect nucleotide is present in the active site. 

 

Figure 6. Proposed sequence of events for the catalytic cycle of DNA pol λ [Foley et al., 2006]. In all panels, the X-ray crystal binary (PDB entry 1XSL) and ternary (PDB entry 1XSN) complexes are green and red, respectively, and intermediate conformations of residues and DNA are blue. Blue arrows represent motion. The correct incoming nucleotide, here dTTP, is shown in all panels with both the catalytic and nucleotide-binding ions (gold spheres) except panel 4. Panel 1 depicts the closing motion of the loop containing ß-strand 8 in the thumb subdomain in response to binding of the correct incoming nucleotide, dTTP (purple); superimposed C_a traces of X-ray crystal binary and ternary are shown. Panel 2 shows the start of Arg514's transition from the binary to the ternary position with the flip of Arg514 from the binary state position away from the DNA as indicated by the arrow. Panel 3 shows the partial flip of Arg517 from the binary to the ternary position. Panel 4 shows the flips of Ile492, Tyr505, and Phe506 from the binary to the ternary state positions. Panel 5 shows the DNA in transition from the binary to the ternary state which occurs after the flips of Ile492, Tyr505, and Phe506. Panel 6 shows the completed transition of the DNA to its ternary position. Panel 7 shows the completed flip of Arg517 from its intermediate position to the ternary position. Panel 8 shows the completed flip of Arg514 from its intermediate position to its ternary position. Panel 9 shows the closed active conformation of the pol λ/DNA complex with substrate (purple) and ions (gold spheres) bound.

 

Computational Investigations into the Conformational and Chemical Pathways in Dpo4

 

The recently discovered low-fidelity Y-family DNA polymerases are particularly interesting because of their lesion-bypassing capacity; namely, they can be loaded onto the replication fork to transverse bulky lesions and rescue replication when the replicative DNA polymerases are stalled.  Sulfolobus solfataricus DNA Polymerase IV (Dpo4), a thermophilic archeal protein capable of translesion synthesis, is one of the few Y-family DNA polymerases crystallized and studied experimentally. The structure of Dpo4 [Ling et al, 2001], like other known DNA polymerases, bears the shape of a hand, containing four protein domains, palm, thumb, fingers, and little finger. The reactive region for nucleotide insertion is located at the palm domain, where the catalytic triad, Asp7, Asp105, and Glu106, as found in other DNA polymerases, coordinate with two metal ions – the nucleotide-binding and catalytic Mg²⁺.

 

Figure 7.  3D structure of Dpo4.

 

The nature of the conformational changes in Dpo4. The high error rate of Y family polymerases in replicating undamaged DNA and their ability to bypass DNA lesions may be partially due to a more open active site (relative to high-fidelity polymerases) that can even accommodate two template bases simultaneously [Ling et al., 2003]. The existence of subtle conformational changes in enzyme subdomains upon binding substrate may be another factor. Indeed, kinetic studies suggest that Y-family’s pol η undergoes a conformational change, possibly in regions far from the active site or even in the DNA; kinetic data [Fiala & Suo, 2004] suggest that the incorporation of a correct incoming nucleotide by Dpo4 before chemistry is limited by the enzyme conformational change, while the incorporation of an incorrect nucleotide is limited by the chemistry step. Combined with the Dpo4/DNA complexes with 8-OxoG [Rechkoblit et al., 2006], comparison of the ternary with each of the two binary structures suggests that a very subtle motion occurs in the little finger before chemistry and the DNA may slide so as to prepare the active site in a desired orientation; this subtle but more complex change relative to pol β may allow the enzyme to accommodate different types of lesions in the active site.

 

Through long-term MD simulations of the ternary Dpo4/DNA/dCTP closed system (with 8-OxoG bound in anti orientation to incoming C) in native-like environments, we have captured significant rigid-body motions (Fig. 8) in the finger and little finger domains that facilitate DNA translocation [Wang et al., 2006].  Compared to the thumb and little finger domains, the catalytic core of Dpo4, particularly the palm domain, is relatively rigid.  The motions of the finger and little finger domains appear to be perpendicular to each other like a propeller twist and push the nascent base pair as well as the DNA duplex away from the active site to make room for the next incoming nucleotide (see Fig. 8). We also find that the DNA translocates by almost a half base pair’s rise, and the template and primer strands move about 2.0 Å and 1.0 Å from the active site, respectively. These observations point to movements of the little finger and the DNA that may help adjust the active site conformation and facilitate the accommodation of bulky lesions [Ling et al., 2003].  Moreover, our simulations suggest that the active site of Dpo4 does not undergo large magnitude conformational changes upon binding of a correct incoming nucleotide as found in higher fidelity pol β.  The catalytic site is also more distorted than that in pol β relative to the reaction-competent conformation [Mildvan, 1997], possibly contributing to the low catalysis efficiency of Dpo4 compared to pol β.

 

Figure 8. Structural comparison of the starting and final Dpo4/DNA complexes in the second simulation after chemistry [Wang et al., 2006]. (A) Superimposition of the simulated structure (light green) in the trajectory after chemistry with metal ions and PPi removed to the ternary crystal structure (light red) according to the palm domains. (B) Enlarged view of the DNA duplexes before (red) and after (green) the simulation. 8-OxoG and dCTP are labeled as OxoG and C, respectively. Black arrow indicates the direction of their movements. (C) Comparison of the LF domains before (light red) and after (light green) simulation to that of the Dbh apo-structure [Silvian et al., 2001] (blue) by superimposing the palm domains.

 

The nucleotidyl transfer reaction in Dpo4. We are currently studying the chemical reaction mechanism of Dpo4 using the coupled QM/MM method. The potential energy surface of several possible reaction pathways in Dpo4 will be delineated by treating the reactive region at the catalytic core quantum mechanically, while the rest of the protein/DNA as well as the solvent environment will be treated using the molecular mechanics method.

 

Emerging Themes and Insights

Our simulation studies of the conformational and chemical pathways in various DNA polymerases reveal key mechanisms used by polymerases to enhance fidelity. Our work indicates that low and high fidelity polymerases function differently in several ways.  Higher fidelity polymerases like pol β exhibit large-scale subdomain motions before and after chemistry.  They also have geometrically restrictive active sites and little tolerance for DNA distortion.  Lower fidelity polymerases have fast subdomain motions (e.g., pol X), no large-scale subdomain motions (e.g., pol λ), and/or extensive DNA motion in their catalytic cycle (e.g., pol λ and Dpo4).  These enzymes are also more tolerant of active site or DNA distortion.  Although the differences are significant and affect polymerase function, our work has also elucidated several similarities among these different polymerases. 

 

The emerging unifying theme is a relationship between cooperative conformational changes and chemical activation that define kinetic checkpoints in nucleotide incorporation and discrimination.  Three distinct avenues can summarize this theme (Fig. 9): conformational change avenue upon substrate binding, prechemistry avenue to evolve the system to the reaction-competent state, and chemistry avenue for the nucleotide transfer. The prechemistry avenue concept may help interpret different polymerase mechanisms through analysis of prechemistry energy barriers. Indeed, data from structural and computational studies of several polymerases (e.g., pol β, pol λ, pol X, and Dpo4) in binary and ternary states emphasize that polymerase conformational changes can vary significantly among polymerases, even within the same family. Thus, while polymerases may appear to lack a unified description based on the subdomain motions alone to explain their wide variation in DNA synthesis and repair fidelities, by invoking the new concept of the prechemistry avenue, we may be able to reconcile some observed differences in the fidelities of various polymerases.

 

Using our new concept of the prechemistry avenue [Radhakrishnan et al., 2006a], we plan to analyze the active sites in various polymerases to understand how critical distances (e.g., between P_a and O3' (primer), catalytic magnesium to O3', and the O1A coordination with the metal ions) regulate the ternary complexes to evolve them differently toward chemistry. Evidence suggests that the evolutionary pressure can lead polymerases to control the rate of the chemical reaction by stabilizing the conformations at different values of the reaction coordinate (in comparison to the transition state), thereby driving the reaction through alternate pathways. Available NMR data already support our hypothesis regarding such conformational rearrangements. Our goal is to find further connections between the conformational rearrangements and cooperative motions that represent key mechanisms used by polymerases to enhance fidelity.  For example, the more distorted active-site geometries of mismatched systems after the conformational closing pathways imply the likely existence of a post-conformational rearrangement of active-site degrees of freedom that could serve as a crucial kinetic checkpoint.

 

Figure 9. Sequential events and corresponding “gates” controlling pol β's fidelity [Radhakrishnan et al., 2006a]: the conformational change avenue, which comprises of Arg192 flip, Phe272 flip, and Arg258 rotation accompanying thumb subdomain closing motions upon incoming nucleotide binding, the pre-chemistry avenue, which involves the stochastic reorganization of the protein catalytic region, particularly the coordinating ligands of the two binding metal ions, and the chemistry avenue, where the incoming nucleotide is finally connected onto the primer terminus and the primer is extended by one residues.

 

Other insights from our work demonstrate important roles for protein residues in substrate binding, conformational transitioning before and after chemistry, and details of polymerase fidelity mechanisms. These hypothesized roles can be tested by both experimental and computational site-directed mutagenesis.  This has already been successfully carried out for pol λ (described above) to define the role of Arg517 on DNA stability and frameshift error generation.  Furthermore, simulations of Dpo4 and pol β DNA ternary complexes associated with an 8-oxoG lesion [Wang et al., 2006; Wang et al., 2007a; Wang & Schlick, 2007b] provide valuable insights into the lesion processing mechanisms in medium and low fidelity DNA polymerases. The little finger domain in Dpo4, unique to known Y-family DNA polymerases, plays a critical role in lesion bypassing because of its capacity to rotate around the DNA major groove and accommodate the bulky lesions. While for pol β, since its active site region is much less flexible than Dpo4, bulky lesions can stall the DNA replication.

 

 

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