DNA molecules consist of two separate strands that spiral around each other to form a structure called the double helix. Each strand contains repeating units, with every unit consisting of a phosphate group and a sugar molecule bound to one of four bases. The two strands are held together by bonds between the bases.

When a cell divides, it needs to make a copy of the DNA, so that each new cell will have an exact replica from the old cell. During this process, the helix unwinds and enzymes called polymerases produce new strands (using the old ones as a template). Each strand is copied by adding new bases one at a time. Every time a new base is added, the polymerases must modify their structures several times. If this process becomes faulty, it can lead to various diseases, including cancer.

Scientist often use a technique called X-ray crystallography to study intermediate structures of frozen polymerase crystals as the enzyme constructs DNA. Yet, to fully understand the mechanisms of DNA synthesis all intermediate structures need to be identified.

Now, Chim, Jackson et al. used a particular method for making frozen polymerase crytals by allowing the enzyme to add new bases in liquid form. The reaction was then frozen and X-ray crystallography was used to take images. This modified method captured different steps in the process and detailed how the enzyme adjusts its structure as it moves along the template strand.

The intermediate structures that Chim, Jackson et al. uncovered may help scientists develop new biotechnologies and medicines. Understanding how polymerases modify their form while making DNA copies could lead to better therapies for diseases in which this process has become faulty, like cancer.

DNA polymerase I (DNAP-I) has long been viewed as the canonical model for DNA synthesis in cells (Lehman et al., 1958). Structural insights into the mechanism of DNA synthesis have been obtained from crystal structures of a thermostable bacterial (Geobacillus stearothermophilus, Bst) DNAP-I large fragment that retains catalytic activity inside the crystal lattice (Johnson et al., 2003; Kiefer et al., 1998). The prevailing mechanism invokes the use of a distinct pre-insertion site, observed in the translocated product of in crystallo catalyzed primer-extension reactions where dNTP substrates are soaked into pre-formed crystals of DNAP-I bound to a primer-template duplex (Figure 1—figure supplement 1)(Johnson et al., 2003; Kiefer et al., 1998). The pre-insertion site is a hydrophobic pocket located between the O and O1 helices of the finger subdomain where the n + 1 templating base resides prior to forming the nascent base pair with the incoming dNTP substrate (Johnson et al., 2003). However, the pre-insertion site has not been witnessed in polymerases with homologous active sites (Eom et al., 1996; Li et al., 1998; Yin and Steitz, 2002), implying that DNAP-I follows a complex enzymatic pathway that contains numerous intermediates, many of which have not yet been observed in protein crystals. Here we report five crystal structures of DNAP-I that capture new conformations for the polymerase translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. Together, these structures provide new insight into the mechanism of DNA synthesis and highlight the dynamic nature of the finger subdomain in the enzyme active site.

Recognizing that in crystallo and solution catalyzed enzymatic reactions can produce different structural results with potentially different functional interpretations (Ehrmann et al., 2017), we chose to investigate the translocated intermediates of DNAP-I using a direct crystallization method that involves solving crystal structures of the enzyme-product complex obtained from primer-extension reactions performed in solution rather than inside the environment of a protein crystal. In these reactions, the starting enzyme-primer-template complex was incubated with solutions of either buffer, dTTP, or dTTP and dATP for 30 min at 37°C. Following primer-extension, the enzyme-product complex was crystallized and cocrystal structures of Bst DNAP-I were solved to resolutions of 1.5 – 2.0 Å (Table 1). This approach was used to obtain high resolution structures of DNAP-I for the starting primer-template complex (n) and two translocated products obtained for the n + 1 and n + 2 nucleotide addition steps using the same primer-template duplex (n) described in previous in crystallo studies (Figure 1a)(Johnson et al., 2003).

Figure 1 with 5 supplements see all

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Structures of the enzyme-primer-template complex (n) before catalysis reflect the initiation step of DNA synthesis. Superposition of the new structure obtained for the initiation step against the previously solved structure reveals that both structures adopt the same active site conformation (Figure 1—figure supplement 2a). This result implies that any structural differences observed between the translocated product of solution and in crystallo catalyzed reactions should be due to the catalysis environment rather than the starting polymerase conformation.

To evaluate the elongation step of DNA synthesis, the translocated products obtained from solution and in crystallo catalyzed primer-extension reactions were compared, both globally and locally within the enzyme active site (Johnson et al., 2003; Kiefer et al., 1998). All of the structures adopt the same overall topology commonly observed for A-family DNA polymerases (Figure 1b). However, careful analysis of the enzyme active site did reveal clear conformational differences between structures obtained from solution-catalyzed reactions versus those obtained from in crystallo catalyzed reactions (Figure 1c,d). The in crystallo catalyzed reactions adopt an active site conformation that is nearly identical to the starting conformation, which represents the initiation step of DNA synthesis (Figure 1—figure supplement 2a). However, the solution catalyzed reactions produce a different active site conformation that binds the duplex in a different position and base pair geometry (Figure 1—figure supplement 2b,c).

Major structural differences are depicted in the 2D interaction maps, which show that the solution catalyzed reactions produce a translocated product with markedly fewer contacts to the phosphodiester linkage, sugar, and nucleobase moieties of the primer-template duplex as compared to the translocated product obtained by in crystallo catalysis (Figure 1—figure supplements 3 and 4, Supplementary file 1a). A particularly striking example of conformational disparity is Tyr⁷¹⁴, a critical active site residue involved in the mechanism of DNA synthesis (Bell et al., 1997; Carroll et al., 1991). In the solution catalyzed structures, Tyr⁷¹⁴ stabilizes the newly formed base pair by stacking above the primer strand, while this residue stacks above the template strand in the in crystallo catalyzed structures (Figure 1c,d). Importantly, the pre-insertion site is not observed in the solution catalyzed reactions due to a kink in the O-helix, which abrogates the O-O1 loop in the finger subdomain (Figure 1d). Absent a hydrophobic pocket, the n + 1 nucleotide in the template strand stacks against Tyr⁷¹⁹ in the O1 helix, which positions the base for a subsequent round of catalysis. The solution catalyzed structures obtained for the n + 1 and n + 2 translocated products adopt identical active site conformations (Figure 1 – figure supplement 2d), which together represent a new intermediate along the DNA replication pathway of Bst DNAP-I.

Next, we examined whether a solution catalyzed conformation could be converted to an in crystallo conformation through a round of in crystallo catalysis. Accordingly, dATP was soaked into a crystal of the n + 1 translocated product obtained by crystallization of a solution catalyzed reaction. Following one cycle of in crystallo catalysis, an n + 2 translocated structure was produced that now contained the pre-insertion site and matched the active site conformation of previous in crystallo results (Figure 1 – figure supplement 2e, f). This observation demonstrates that in crystallo catalysis favors an active site conformation that contains the pre-insertion site, as the same active site conformation is obtained from two different starting points.

Interestingly, the translocated product obtained from the set of solution catalyzed reactions is similar to known Bst DNAP-I structures solved with duplexes that contain damaged DNA intermediates and active site mutations (Figure 1—figure supplement 5, Supplementary file 1b). These structures were previously thought to contain a distorted active site conformation due to the position of Tyr⁷¹⁴ relative to its conformation in the in crystallo catalysis structures (Gehrke et al., 2013; Johnson and Beese, 2004; Wang et al., 2012). However, given the homology of these structures to the translocated product of solution catalyzed reactions, we postulate that Tyr⁷¹⁴ functions as a regulatory checkpoint in the mechanism of DNA synthesis by evaluating the geometry of the newly formed base pair.

Next, we wondered whether the mechanism of DNAP-I included the formation of a pre-insertion complex, which is a ternary structure different from the previously discussed pre-insertion site observed in the binary structure of in crystallo catalyzed primer-extension reactions. Previously, Wu and colleagues solved the ternary structure of a mutant version of Bst DNAP-I bound to an incoming dATP substrate (Miller et al., 2015). Although that structure was originally described as an open ternary complex, presumably to avoid confusion with the pre-insertion site, it resembles the pre-insertion complex first observed in Klentaq1 (Li et al., 1998). The key difference between the open ternary and pre-insertion complex is whether the incoming nucleotide is paired opposite the templating base or an active site residue (Doublié et al., 1998; Yin and Steitz, 2004). Since the structure by Wu and colleagues shows the incoming substrate paired opposite Tyr⁷¹⁴, it should be considered a pre-insertion complex.

We demonstrated that the wild-type polymerase is also capable of forming a pre-insertion complex by solving the ternary structure of the enzyme bound to the non-hydrolyzable analog, dAMPNPP. The resulting structure (Figure 2) closely resembles the mutant Bst polymerase structure determined by Wu and colleagues and shows Tyr⁷¹⁴ paired opposite the incoming nucleotide (Miller et al., 2015). Although the phosphate tail shows nearly 100% occupancy, the sugar and nucleobase moieties are flexible, which is consistent with the dynamic properties of the incoming nucleotide in an open polymerase conformation. Nevertheless, the structure shows that the incoming nucleotide is stabilized by polar contacts to the negatively charged triphosphate moiety. These observations demonstrate that Bst DNAP-I adopts a pre-insertion complex similar to other A-family DNA polymerases (Rothwell and Waksman, 2005), which clarifies an important step in the mechanism of DNA synthesis.

Figure 2

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Based on the structures reported here, we propose a revised mechanism for DNA synthesis by DNA polymerase I. The catalytic cycle consists of four key steps that derive from high resolution structures of Bst DNAP-I and its homolog T7 RNA polymerase (Figure 3). Starting from the newly determined post-translocation complex, the polymerase undergoes a conformation change to adopt the pre-insertion complex with an incoming nucleotide paired opposite Tyr⁷¹⁴ in the enzyme active site. This conformational change involves release of the n + 1 templating base from its stacking interaction with Tyr⁷¹⁹ in the O1 helix and the repositioning of Tyr⁷¹⁴ in the enzyme active site. The enzyme then undergoes a more significant conformational change to adopt the closed ternary complex (Johnson et al., 2003), which defines the pre-catalytic state of the enzyme. Immediately following phosphodiester bond formation, the enzyme adopts a post-catalytic complex in which the primer has been extended by one nucleotide (Yin and Steitz, 2004). The enzyme then translocates to the next position on the template to initiate another cycle of nucleotide addition.

Figure 3

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In summary, we present crystal structures of DNA polymerase I that capture the translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. We suggest that these new structures, along with previously solved structures obtained by in crystallo catalysis, highlight the dynamic nature of the finger subdomain in the enzyme active site. Together, the new and existing structures expand our understanding of the mechanism of DNA synthesis by capturing important intermediates in a complicated reaction pathway.

Coordinates and structure factors have been deposited in the PDB with the accession codes: 6DSU, 6DSV, 6DSW, 6DSX, and 6DSY.

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Thank you for submitting your article "Crystal Structures of DNA Polymerase I Capture the Physiological Intermediates of Translocation and Pre-Insertion" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by John Kuriyan as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

This manuscript from Chim et al. describes the results of a crystallographic characterization of the Bst DNA polymerase I large fragment, a model enzyme for understanding the A-family DNA polymerases. The authors describe novel structures, compared with those in previous publications in the field and deposited in the PDB. Instead of relying on use of an in crystallo reaction set up toward following the reaction cycle, the authors used "direct" crystallization of complexes formed in solution. The crystal structures obtained in this work are generally similar to the crystal structures published by the group of Lorena Beese (Johnson et al., 2003; Kiefer et al., 1998), using the identical DNA polymerase and DNA substrates. In the original work, the different structures were obtained by extending a DNA substrate by one or two nucleotides in crystals. In the current work, the authors reproduce identical structures by performing the same in-crystal extension of the DNA substrate. The authors also present two new structures that are the results of performing the extension in solution before crystallization. These new structures differ from the in-crystal structures in that the template base that is to be paired with the incoming nucleotide (n+1 base) is not bound to the so-called pre-insertion site but positioned on the other side of a helix that borders the pre-insertion site.

In summary, the authors present structures of reaction intermediates in the DNA polymerase cycle that have not been observed previously. The results emphasize the point of dynamics in the fingers domain as the enzyme transitions from one step to the next in the reaction cycle.

We agree that this work is important, and potentially suitable for publication in eLife, because of the fundamental importance of DNA replication mechanisms.

We have serious concerns, however, with the way that the overall import of the work is currently presented, in terms of its potential "physiological relevance". All structural analyses are results of artifacts of the solution or crystal conditions, and it is likely that conformations that are stable enough to be trapped in one way or the other represent intermediates or transitions on an underlying free-energy surface that are inadequately sampled by current experimental methods and our inability to mimic cellular conditions. In particular, the authors argue that their new structures are "physiologically relevant", while those obtained previously through in-crystal extension are not. The authors provide no evidence to support this statement.

Their first argument is that the "pre-insertion site is inconsistent with the fast rate of Bst DNAP-I (~200nt/s)" (Introduction). Yet the authors do not explain why this would be inconsistent. They also claim that the binding of the n+1 base in the pre-insertion site "has not been witnessed in polymerases with homologous active sites" (Introduction). However, one of the referenced structures is without DNA, one is an RNA polymerase with a DNA-RNA hybrid substrate, and two of these are from a different species (Thermus aquaticus), both of which did not include a primer extension step. In contrast, one paper (Golosov, 2010) describes a molecular dynamics analysis that is completely consistent with the binding of the n+1 base into the pre-insertion site.

Curiously, the conformation of the DNA template observed in their new structure has been observed before, but only in structures "that contain damaged DNA intermediates and active site mutations" (Results and Discussion section). This might even argue against the new structures representing the "physiologically relevant state", at least for normal base pairs.

It is possible that both sets of structures could represent intermediates in a complex DNA extension cycle that contains many different intermediate structures. We do not feel that it is necessary to diminish the importance of previous contributions to gain value for this new work, which we find interesting in its own right.

We are prepared to consider a revised manuscript that takes into account the points made below. It is stressed, however, that the editors and the reviewers require that the Title and the language of the paper be changed so that a dichotomy, unsupported by additional data, not be presented between "physiologically relevant" and "physiologically irrelevant". No new experiments are requested.

Essential Revisions:

As noted above, change the Title of the paper to not imply physiological relevance or irrelevance.

It seems that the authors have a strong bias against the original, in-crystal extended structures, a bias that is voiced throughout the paper without much clarification. Please remove or modify these speculations in light of the comments made above. Some of these are pointed out below:

"…the pre-insertion site may be a constraint of the crystal lattice and not a relevant intermediate in the DNA synthesis pathway." (Introduction)

"Recognizing that in crystallo and solution catalyzed enzymatic reactions can produce different structural results with different functional interpretations," (Introduction)

"This work provides a telltale illustration of the problems that can arise when soaking substrates into preformed crystals of enzymes that undergo significant conformational changes (Ehrmann et al., 2017). To avoid such problems, we suggest validating important soaking results with cocrystallization."

Introduction, “[…] the pre-insertion site is inconsistent with the fast rate of Bst DNAP-I (~200 nt/sc) […]”: the inconsistency asserted is not clear and is not explained. These comments undercut the manuscript and are not meaningful as written.

In summary, the manuscript should be revised to highlight the impact of the present work without repudiating the significance of previous work.

We agree that this work is important, and potentially suitable for publication in eLife, because of the fundamental importance of DNA replication mechanisms.

We have serious concerns, however, with the way that the overall import of the work is currently presented, in terms of its potential "physiological relevance". All structural analyses are results of artifacts of the solution or crystal conditions, and it is likely that conformations that are stable enough to be trapped in one way or the other represent intermediates or transitions on an underlying free-energy surface that are inadequately sampled by current experimental methods and our inability to mimic cellular conditions. In particular, the authors argue that their new structures are "physiologically relevant", while those obtained previously through in-crystal extension are not. The authors provide no evidence to support this statement.

We agree with the reviewers that, without additional experimental evidence, the claim of physiological relevance or irrelevance by any set of crystal structures is premature and unjustified. In the revised manuscript, we have removed all discussion of physiological relevance and presented all structural intermediates as a continuum of a complex DNA synthesis pathway.

Their first argument is that the "pre-insertion site is inconsistent with the fast rate of Bst DNAP-I (~200nt/s)" (Introduction). Yet the authors do not explain why this would be inconsistent. They also claim that the binding of the n+1 base in the pre-insertion site "has not been witnessed in polymerases with homologous active sites" (Introduction). However, one of the referenced structures is without DNA, one is an RNA polymerase with a DNA-RNA hybrid substrate, and two of these are from a different species (Thermus aquaticus), both of which did not include a primer extension step. In contrast, one paper (Golosov, 2010) describes a molecular dynamics analysis that is completely consistent with the binding of the n+1 base into the pre-insertion site.

We removed the claim that the pre-insertion site is inconsistent with the fast rate of Bst DNAP-I. However, we chose to retain the statement that the pre-insertion site has not been witnessed in polymerases with homologous active sites, as this sentence provides justification for the current work. In addition to changes to the text, references were provided to support the claims.

Curiously, the conformation of the DNA template observed in their new structure has been observed before, but only in structures "that contain damaged DNA intermediates and active site mutations" (Results and Discussion section). This might even argue against the new structures representing the "physiologically relevant state", at least for normal base pairs.

We chose to keep this paragraph in the manuscript as it describes a key observation that provides support for Tyr⁷¹⁴ as a possible regulatory checkpoint. We do not feel that this paragraph diminishes previous work, as all assertions of physiological relevance or irrelevance have been removed from the text.

It is possible that both sets of structures could represent intermediates in a complex DNA extension cycle that contains many different intermediate structures. We do not feel that it is necessary to diminish the importance of previous contributions to gain value for this new work, which we find interesting in its own right.

The entire manuscript was revised to state that all intermediates identified in the current study and previous work represent individual steps in a complex DNA extension pathway.

We are prepared to consider a revised manuscript that takes into account the points made below. It is stressed, however, that the editors and the reviewers require that the Title and the language of the paper be changed so that a dichotomy, unsupported by additional data, not be presented between "physiologically relevant" and "physiologically irrelevant". No new experiments are requested.

Essential Revisions:

As noted above, change the Title of the paper to not imply physiological relevance or irrelevance.

We have changed the Title and revised the Abstract to remove any discussion of physiological relevance or irrelevance.

It seems that the authors have a strong bias against the original, in-crystal extended structures, a bias that is voiced throughout the paper without much clarification. Please remove or modify these speculations in light of the comments made above. Some of these are pointed out below:

"…the pre-insertion site may be a constraint of the crystal lattice and not a relevant intermediate in the DNA synthesis pathway." (Introduction)

This sentence was removed from the text and surrounding sentences were modified.

"Recognizing that in crystallo and solution catalyzed enzymatic reactions can produce different structural results with different functional interpretations," (Introduction).

In the absence of any discussion of physiological relevance, we feel this sentence remains true and does not diminish the impact of previous work. However, we have included the word ‘potentially’ in the sentence to soften the language.

"This work provides a telltale illustration of the problems that can arise when soaking substrates into preformed crystals of enzymes that undergo significant conformational changes (Ehrmann et al., 2017). To avoid such problems, we suggest validating important soaking results with cocrystallization."

These sentences were removed from the text and the summary paragraph was re-written to be consistent with the revised text.

Introduction, “[…] the pre-insertion site is inconsistent with the fast rate of Bst DNAP-I (~200 nt/sc) […]”: the inconsistency asserted is not clear and is not explained. These comments undercut the manuscript and are not meaningful as written.

These lines were removed from the manuscript.

In summary, the manuscript should be revised to highlight the impact of the present work without repudiating the significance of previous work.

We have carefully rewritten the entire manuscript to present all structural intermediates, new and known, as a continuum of a complex DNA synthesis pathway.

Author details

1. Nicholas Chim

   Departments of Pharmaceutical Sciences, University of California, Irvine, California

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing, Conceived of the project and designed the experiments, Performed all the experiments that led to Bst DNAP-I crystals, Collected and processed all X-ray diffraction data sets

   Contributed equally with

   Lynnette N Jackson

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2274-5305

2. Lynnette N Jackson

   Departments of Pharmaceutical Sciences, University of California, Irvine, California

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing, Performed all the experiments that led to Bst DNAP-I crystals, Collected and processed all X-ray diffraction data sets

   Contributed equally with

   Nicholas Chim

   Competing interests

   No competing interests declared

3. Anh M Trinh

   Departments of Pharmaceutical Sciences, University of California, Irvine, California

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing, Performed all the experiments that led to Bst DNAP-I crystals

   Competing interests

   No competing interests declared

4. John C Chaput

   1. Departments of Pharmaceutical Sciences, University of California, Irvine, California
   2. Department of Chemistry, University of California, Irvine, California
   3. Department of Molecular Biology and Biochemistry, University of California, Irvine, California

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing, Conceived of the project and designed the experiments

   For correspondence

   jchaput@uci.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1393-135X

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