Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms

DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase–DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s⁻¹, much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.     

Motions of the fingers subdomain of klentaq1 are fast and not rate limiting: implications for the molecular basis of fidelity in DNA polymerases

Various kinetic studies on nucleotide incorporation by DNA polymerases have established that a rate-limiting step occurs that is crucial in the mechanism of discrimination between correct versus incorrect nucleotide. Crystallographic studies have indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. However, there is no direct evidence to support this hypothesis. In order to investigate whether or not the open-to-closed conformational transition affecting the fingers subdomain is rate limiting, we have developed a fluorescence resonance energy transfer (FRET) system, which monitors motions of the fingers subdomain. We establish that the closing of the fingers subdomain is significantly faster than the kinetically determined rate-limiting step. We propose that the rate-limiting step occurs after the closing of the fingers subdomain and is caused by local reorganization events in the active site.     

Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue

DNA polymerases discriminate between correct and incorrect nucleotide substrates during a "nonchemical" step that precedes phosphodiester bond formation in the enzymatic cycle of nucleotide incorporation. Despite the importance of this process in polymerase fidelity, the precise nature of the molecular events involved remains unknown. Here we report a fluorescence resonance energy transfer (FRET) system that monitors conformational changes of a polymerase-DNA complex during selection and binding of nucleotide substrates. This system utilizes the fluorescent base analogue 1,3-diaza-2-oxophenothiazine (tC) as the FRET donor and Alexa-555 (A555) as the acceptor. The tC donor was incorporated within a model DNA primer/template in place of a normal base, adjacent to the primer 3' terminus, while the A555 acceptor was attached to an engineered cysteine residue (C751) located in the fingers subdomain of the Klenow fragment (KF) polymerase. The FRET efficiency increased significantly following binding of a correct nucleotide substrate to the KF-DNA complex, showing that the fingers had closed over the active site. Fluorescence anisotropy titrations utilizing tC as a reporter indicated that the DNA was more tightly bound by the polymerase under these conditions, consistent with the formation of a closed ternary complex. The rate of the nucleotide-induced conformational transition, measured in stopped-flow FRET experiments, closely matched the rate of correct nucleotide incorporation, measured in rapid quench-flow experiments, indicating that the conformational change was the rate-limiting step in the overall cycle of nucleotide incorporation for the labeled KF-DNA system. Taken together, these results indicate that the FRET system can be used to probe enzyme conformational changes that are linked to the biochemical function of DNA polymerase.     

phi 29 DNA polymerase residue Leu384, highly conserved in motif B of eukaryotic type DNA replicases,is involved in nucleotide insertion fidelity

Replicative DNA polymerases achieve insertion fidelity by geometric selection of a complementary nucleotide followed by induced fit: movement of the fingers subdomain toward the active site to enclose the incoming and templating nucleotides generating a binding pocket for the nascent base pair. Several residues of motif B of DNA polymerases from families A and B, localized in the fingers subdomain, have been described to be involved in template/primer binding and dNTP selection. Here we complete the analysis of this motif, which has the consensus "KLX2NSXYG" in DNA polymerases from family B, characterized by mutational analysis of conserved leucine, Leu384 of phi 29 DNA polymerase. Mutation of Leu384 into Arg resulted in a phi 29 DNA polymerase with reduced nucleotide insertion fidelity during DNA-primed polymerization and protein-primed initiation reactions. However, the mutation did not alter the intrinsic affinity for the different dNTPs, as shown in the template-independent terminal protein-deoxynucleotidylation reaction. We conclude that Leu384 of phi 29 DNA polymerase plays an important role in positioning the templating nucleotide at the polymerization active site and in controlling nucleotide insertion fidelity. This agrees with the localization of the corresponding residue in the closed ternary complexes of family A and family B DNA polymerases, contributing to form the binding pocket for the nascent base pair. As an additional effect, mutant polymerase L384R was strongly reduced in DNA binding, resulting in reduced processivity during polymerization.     

Solution structure of a viral DNA polymerase X and evidence for a mutagenic function.

The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. Here we report the solution structure of Pol X. The structure, unique within the realm of nucleotide polymerases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is important for DNA binding in other polymerases, we show that Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may contribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator.     

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β: THE CRITICAL ROLE OF FINGERS DOMAIN MOVEMENTS AND A NOVEL NON-COVALENT STEP DURING NUCLEOTIDE SELECTION*

During DNA repair, DNA polymerase β (Pol β) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol β fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol β. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.     

A pre-equilibrium before nucleotide binding limits fingers subdomain closure by Klentaq1

Numerous studies have been undertaken to establish the mechanism of dNTP binding and template-directed incorporation by DNA polymerases. It has been established by kinetic experiments that a rate-limiting step, crucial for dNTP selection, occurs before chemical bond formation. Crystallographic studies indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. In previous studies, we established a fluorescence resonance energy transfer system to monitor the open-to-closed transition in the fingers subdomain of Klentaq1. By comparing the rates of the fingers subdomain closure with that of the rate-limiting step for Klentaq1, we showed that fingers subdomain motion was significantly faster than the rate-limiting step. We have now used this system to characterize DNA binding as well as to complete a more extensive characterization of incorporation of all four dNTPs. The data indicate that DNA binding occurs by a two-step association and that dissociation of the DNA is significantly slower in the case of the closed ternary complex. The data for nucleotide incorporation indicate a step occurring before dNTP binding, which differs for all four nucleotides. As the only difference between the (E x p/t) complexes is the templating base, it would suggest an important role for the templating base in initial ground state selection.     

Structural insights into DNA polymerase beta deterrents for misincorporation support an induced-fit mechanism for fidelity

DNA polymerases generally select the correct nucleotide from a pool of structurally similar molecules to preserve Watson-Crick base-pairing rules. We report the structure of DNA polymerase beta with DNA mismatches situated in the polymerase active site. This was achieved by using nicked product DNA that traps the mispair (template-primer, A-C or T-C) in the nascent base pair binding pocket. The structure of each mispair complex indicates that the bases do not form hydrogen bonds with one another, but form a staggered arrangement where the bases of the mispair partially overlap. This prevents closure/opening of the N subdomain that is believed to be required for catalytic cycling. The partially open conformation of the N subdomain results in distinct hydrogen bonding networks that are unique for each mispair. These structures define diverse molecular aspects of misinsertion that are consistent with the induced-fit model for substrate specificity.     

The Rate-limiting Step of DNA Synthesis by DNA Polymerase Occurs in the Fingers-closed Conformation

DNA polymerases maintain genomic integrity by copying DNA with high fidelity, part of which relies on the polymerase fingers opening-closing transition, a series of conformational changes during the DNA synthesis reaction cycle. Fingers opening and closing has been challenging to study, mainly due to the need to synchronise molecular ensembles. We previously studied fingers opening-closing on single polymerase-DNA complexes using single-molecule FRET; however, our work was limited to pre-chemistry reaction steps. Here, we advance our analysis to extensible substrates, and observe DNA polymerase (Pol) conformational changes across the entire DNA polymerisation reaction in real-time, gaining direct access to an elusive post-chemistry step rate-limiting for DNA synthesis. Our results showed that Pol adopts the fingers-closed conformation during polymerisation, and that the post-chemistry rate-limiting step occurs in the fingers-closed conformation. We found that fingers-opening in the Pol-DNA binary complex in the absence of polymerisation is slow (～5.3 s^?1), and comparable to the rate of fingers-opening after polymerisation (3.4 s^?1); this indicates that the fingers-opening step itself could be largely responsible for the slow post-chemistry step, with the residual rate potentially accounted for by pyrophosphase release. We also observed that DNA chain-termination of the 3′ end of the primer increases substantially the rate of fingers-opening in the Pol-DNA binary complex (5.3 → 29 s^?1), demonstrating that the 3′-OH residue is important for the kinetics of fingers conformational changes. Our observations offer mechanistic insight and tools to offer mechanistic insight for all nucleic acid polymerases.     

In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism

The African swine fever virus DNA polymerase X (pol X), a member of the X family of DNA polymerases, is thought to be involved in base excision repair. Kinetics data indicate that pol X catalyzes DNA polymerization with low fidelity, suggesting a role in viral mutagenesis. Though pol X lacks the fingers domain that binds the DNA in other members of the X family, it binds DNA tightly. To help interpret details of this interaction, molecular dynamics simulations of free pol X at different salt concentrations and of pol X bound to gapped DNA, in the presence and in the absence of the incoming nucleotide, are performed. Anchors for the simulations are two NMR structures of pol X without DNA and a model of one NMR structure plus DNA and incoming nucleotide. Our results show that, in its free form, pol X can exist in two stable conformations that interconvert to one another depending on the salt concentration. When gapped double stranded DNA is introduced near the active site, pol X prefers an open conformation, regardless of the salt concentration. Finally, under physiological conditions, in the presence of both gapped DNA and correct incoming nucleotide, and two divalent ions, the thumb subdomain of pol X undergoes a large conformational change, closing upon the DNA. These results predict for pol X a substrate-induced conformational change triggered by the presence of DNA and the correct incoming nucleotide in the active site, as in DNA polymerase beta. The simulations also suggest specific experiments (e.g., for mutants Phe-102Ala, Val-120Gly, and Lys-85Val that may reveal crucial DNA binding and active-site organization roles) to further elucidate the fidelity mechanism of pol X.     

Interaction of DNA polymerase I (Klenow fragment) with the single-stranded template beyond the site of synthesis

Cocrystal structures of DNA polymerases from the Pol I (or A) family have provided only limited information about the location of the single-stranded template beyond the site of nucleotide incorporation, revealing contacts with the templating position and its immediate 5' neighbor. No structural information exists for template residues more remote from the polymerase active site. Using a competition binding assay, we have established that Klenow fragment contacts at least the first four unpaired template nucleotides, though the quantitative contribution of any single contact is relatively small. Photochemical cross-linking indicated that the first unpaired template base beyond the primer terminus is close to Y766, as expected, and the two following template bases are close to F771 on the surface of the fingers subdomain. We have constructed point mutations in the region of the fingers subdomain implicated by these experiments. Cocrystal structures of family A DNA polymerases predict contacts between the template strand and S769, F771, and R841, and our DNA binding assays provide evidence for the functional importance of these contacts. Overall, the data are most consistent with the template strand following a path over the fingers subdomain, close to the side chain of R836 and a neighboring cluster of positively charged residues.     

Crystal structure of the catalytic core of human DNA polymerase kappa

We present the crystal structure of the catalytic core of human DNA polymerase kappa (hPolkappa), the first structure of a human Y-family polymerase. hPolkappa is implicated in the proficient extension of mispaired primer termini on undamaged DNAs, and in the extension step of lesion bypass. The structure reveals a stubby "fingers" subdomain, which despite its small size appears to be tightly restrained with respect to a putative templating base. The structure also reveals a novel "thumb" subdomain that provides a basis for the importance of the N-terminal extension unique to hPolkappa. And, most surprisingly, the structure reveals the polymerase-associated domain (PAD) juxtaposed on the dorsal side of the "palm" subdomain, as opposed to the fingers subdomain. Together, these properties suggest that the hPolkappa active site is constrained at the site of the templating base and incoming nucleotide, but the polymerase is less constrained following translocation of the lesion.     

Kinetic analysis of nucleotide incorporation by mammalian DNA polymerase delta

The kinetics of nucleotide incorporation into 24/36-mer primer/template DNA by purified fetal calf thymus DNA polymerase (pol) delta was examined using steady-state and pre-steady-state kinetics. The role of the pol delta accessory protein, proliferating cell nuclear antigen (PCNA), on DNA replication by pol delta was also examined by kinetic analysis. The steady-state parameter k(cat) was similar for pol delta in the presence and absence of PCNA (0.36 and 0.30 min(-1), respectively); however, the K(m) for dNTP was 20-fold higher in the absence of PCNA (0.067 versus 1.2 microm), decreasing the efficiency of nucleotide insertion. Pre-steady-state bursts of nucleotide incorporation were observed for pol delta in the presence and absence of PCNA (rates of polymerization (k(pol)) of 1260 and 400 min(-1), respectively). The reduction in polymerization rate in the absence of PCNA was also accompanied by a 2-fold decrease in burst amplitude. The steady-state exonuclease rate of pol delta was 0.56 min(-1) (no burst, 10(3)-fold lower than the rate of polymerization). The small phosphorothioate effect of 2 for correct nucleotide incorporation into DNA by pol delta.PCNA indicated that the rate-limiting step in the polymerization cycle occurs prior to phosphodiester bond formation. A K(d)(dNTP) value of 0.93 microm for poldelta.dNTP binding was determined by pre-steady-state kinetics. A 5-fold increase in K(d)(DNA) for the pol delta.DNA complex was measured in the absence of PCNA. We conclude that the major replicative mammalian polymerase, pol delta, exhibits kinetic behavior generally similar to that observed for several prokaryotic model polymerases, particularly a rate-limiting step following product formation in the steady state (dissociation of oligonucleotides) and a rate-limiting step (probably conformational change) preceding phosphodiester bond formation. PCNA appears to affect pol delta replication in this model mainly by decreasing the dissociation of the polymerase from the DNA.     

Insights into DNA replication: the crystal structure of DNA polymerase B1 from the archaeon Sulfolobus solfataricus

To minimize the large number of mispairs during genome duplication owing to the large amount of DNA to be synthesized, many replicative polymerases have accessory domains with complementary functions. We describe the crystal structure of replicative DNA polymerase B1 from the archaeon Sulfolobus solfataricus. Comparison between other known structures indicates that although the protein is folded into the typical N-terminal, editing 3'-5'exonuclease, and C-terminal right-handed polymerase domains, it is characterized by the unusual presence of two extra alpha helices in the N-terminal domain interacting with the fingers helices to form an extended fingers subdomain, a structural feature that can account for some functional features of the protein. We explore the structural basis of specific lesion recognition, the initial step in DNA repair, describing how the N-terminal subdomain pocket of archaeal DNA polymerases could allow specific recognition of deaminated bases such as uracil and hypoxanthine in addition to the typical DNA bases.     

Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7

Ultraviolet-induced DNA damage poses a lethal block to replication. To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD). When the 3' thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation. When the 5' thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3' base of the primer, while the fingers are in a closed conformation. These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.     

Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9 degrees N-7

The 2.25 A resolution crystal structure of a pol alpha family (family B) DNA polymerase from the hyperthermophilic marine archaeon Thermococcus sp. 9 degrees N-7 (9 degrees N-7 pol) provides new insight into the mechanism of pol alpha family polymerases that include essentially all of the eukaryotic replicative and viral DNA polymerases. The structure is folded into NH(2)- terminal, editing 3'-5' exonuclease, and polymerase domains that are topologically similar to the two other known pol alpha family structures (bacteriophage RB69 and the recently determined Thermococcus gorgonarius), but differ in their relative orientation and conformation.The 9 degrees N-7 polymerase domain structure is reminiscent of the "closed" conformation characteristic of ternary complexes of the pol I polymerase family obtained in the presence of their dNTP and DNA substrates. In the apo-9 degrees N-7 structure, this conformation appears to be stabilized by an ion pair. Thus far, the other apo-pol alpha structures that have been determined adopt open conformations. These results therefore suggest that the pol alpha polymerases undergo a series of conformational transitions during the catalytic cycle similar to those proposed for the pol I family. Furthermore, comparison of the orientations of the fingers and exonuclease (sub)domains relative to the palm subdomain that contains the pol active site suggests that the exonuclease domain and the fingers subdomain of the polymerase can move as a unit and may do so as part of the catalytic cycle. This provides a possible structural explanation for the interdependence of polymerization and editing exonuclease activities unique to pol alpha family polymerases.We suggest that the NH(2)-terminal domain of 9 degrees N-7 pol may be structurally related to an RNA-binding motif, which appears to be conserved among archaeal polymerases. The presence of such a putative RNA- binding domain suggests a mechanism for the observed autoregulation of bacteriophage T4 DNA polymerase synthesis by binding to its own mRNA. Furthermore, conservation of this domain could indicate that such regulation of pol expression may be a characteristic of archaea. Comparion of the 9 degrees N-7 pol structure to its mesostable homolog from bacteriophage RB69 suggests that thermostability is achieved by shortening loops, forming two disulfide bridges, and increasing electrostatic interactions at subdomain interfaces.     

DNA stabilization at the Bacillus subtilis PolX core��a binding model to coordinate polymerase, AP-endonuclease and 3��-5�� exonuclease activities

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3′-5′ exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs.