polymerase-DNA Complex Research Articles

Interplay between DNA Polymerase and Proliferating Cell Nuclear Antigen Switches Off Base Excision Repair of Uracil and Hypoxanthine during Replication in Archaea

Archaeal family-B DNA polymerases bind tightly to uracil and hypoxanthine (the deamination products of cytosine and adenine), resulting in profound inhibition of DNA replication. Investigation of the mechanism of inhibition, using single-turnover kinetics with polymerase in excess of DNA, indicated that deoxy-NTPs were efficiently bound to the polymerase–DNA complex but very poorly incorporated into the extending chain. Addition of the processivity factor proliferating cell nuclear antigen (PCNA) resulted in increased affinity of the polymerase for all primer–templates, producing extremely tight complexes when uracil ( K d = 16 pM) or hypoxanthine ( K d = 65 pM) was present. Analytical ultracentrifugation confirmed the stability of these complexes and revealed a polymerase/PCNA/DNA stoichiometry of 1:1:1. However, PCNA had no influence on the ability of the polymerase to read through uracil and hypoxanthine, the same kinetic parameters being observed with or without the processivity factor. The specificity constants determined using single-turnover kinetics showed that uracil and hypoxanthine slowed the polymerase by factors of ∼ 5000 and 3000, respectively. Uracil and hypoxanthine are removed from DNA by base excision repair, initiated by uracil-DNA glycosylase and endonuclease V, respectively. Both enzymes are profoundly inhibited by the simultaneous binding of both PCNA and polymerase to primer–templates, with polymerase alone being much less effective. Thus, when the PCNA–polymerase complex encounters uracil/hypoxanthine in DNA templates, base excision repair is switched off, protecting the complex from a repair pathway that is dangerous in the context of single-stranded DNA formed during replication.

Time-resolved footprinting for the study of the structural dynamics of DNA–protein interactions

Transcription is often regulated at the level of initiation by the presence of transcription factors or nucleoid proteins or by changing concentrations of metabolites. These can influence the kinetic properties and/or structures of the intermediate RNA polymerase-DNA complexes in the pathway. Time-resolved footprinting techniques combine the high temporal resolution of a stopped-flow apparatus with the specific structural information obtained by the probing agent. Combined with a careful quantitative analysis of the evolution of the signals, this approach allows for the identification and kinetic and structural characterization of the intermediates in the pathway of DNA sequence recognition by a protein, such as a transcription factor or RNA polymerase. The combination of different probing agents is especially powerful in revealing different aspects of the conformational changes taking place at the protein-DNA interface. For example, hydroxyl radical footprinting, owing to their small size, provides a map of the solvent-accessible surface of the DNA backbone at a single nucleotide resolution; modification of the bases using potassium permanganate can reveal the accessibility of the bases when the double helix is distorted or melted; cross-linking experiments report on the formation of specific amino acid-DNA contacts, and DNase I footprinting results in a strong signal-to-noise ratio from DNA protection at the binding site and hypersensitivity at curved or kinked DNA sites. Recent developments in protein footprinting allow for the direct characterization of conformational changes of the proteins in the complex.

Fingers-Closing and Other Rapid Conformational Changes in DNA Polymerase I (Klenow Fragment) and Their Role in Nucleotide Selectivity

We have developed a FRET-based assay for the fingers-closing conformational transition that occurs when a binary complex of DNA polymerase I (Klenow fragment) with a primer-template binds a complementary dNTP and have used this and other fluorescence assays to place the fingers-closing step within the reaction pathway. Because the rate of fingers-closing was substantially faster than the rate of nucleotide incorporation measured in chemical quench experiments, fingers-closing cannot be the rate-limiting prechemistry step defined by earlier kinetic studies. Experiments using Ca (2+) instead of Mg (2+) as the metal cofactor suggest instead that the prechemistry step may involve a change in metal ion occupancy at the polymerase active site. The use of ribonucleotide substrates shows there is a base discriminating step that precedes fingers-closing. This earlier step, detected by 2-AP fluorescence, is promoted by complementary nucleotides (ribo- as well as deoxyribo-) but is blocked by mismatches. The complementary rNTP blocks the subsequent fingers-closing step. Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.

Solution structures of 2 : 1 and 1 : 1 DNA polymerase–DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme–DNA complexes of rat DNA polymerase β (Pol β) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol β–DNA complex, whereas only 1 : 1 Pol X–DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol β–DNA and 1 : 1 Pol X–DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5′-dRP lyase domain of the second Pol β molecule with the active site of the 1 : 1 Pol β–DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5′-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5′-dRP lyase reaction in the crystal structures of the closed ternary enzyme–DNA–dNTP complexes, we propose that the asymmetric 2 : 1 Pol β–DNA complex enhances the function of Pol β.

New targets for new anti-herpes drugs

Although infections are often subclinical, herpes simplex virus (HSV) can cause mild to severe diseases, especially in immunocompromised patients. There are few drugs licensed for the treatment of HSV infections. Most target the viral DNA polymerase, such as acyclovir that remains the reference treatment some thirty years after its discovery! Extensive clinical use of this drug has led to the emergence of resistant strains, mainly in immunocompromised patients, these infections can be managed with only two drugs, foscarnet and cidofovir, both much more toxic than acyclovir. This highlights the crucial need for the development of new anti-herpes drugs that can inhibit infection by both wild-type viruses and drug-resistant strains. Over the last few years, significant efforts have been made to set up a range of strategies for the identification of potential new antiviral drugs. One alternative is to develop drugs with different mechanisms of action. The present article reviews potential viral and cellular targets that are now known to be involved in HSV multiplication and for which specific inhibitors with anti-HSV activity, at least in cell culture, have been identified. These drugs inhibit viral proteins involved in viral replication (DNA polymerase, ribonucleotide reductase or helicase-primase complex). Other drugs acting on cellular proteins needed for viral replication have also been described; these drugs are targetting cyclin-dependent kinases or the polyamine biosynthetic pathway.

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.

A Mg2+‐induced conformational switch rendering a competent DNA polymerase catalytic complex

The structural and dynamical changes occurring before nucleotide addition were studied using molecular dynamics (MD) simulations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexes containing one or two Mg2+ ions in the presence of dNTP. Our models revealed that the formation of a catalytically competent DNA polymerase complex required subtle rearrangements at the catalytic site A, which occurred only when an Mg2+ ion was bound. This model has been validated using pre-steady-state kinetics to show that free Mg2+ is necessary to obtain a catalytically competent polymerase. Kinetic studies carried out with Be2+ as a cofactor permitted the functional discrimination between metal sites A and B. At low concentrations, Be2+ increased the catalytic efficiency of the polymerase, while at higher concentrations, it competed with Mg2+ for binding to site A, and inhibited DNA polymerization. In agreement with experimental data, MD simulations revealed that the catalytic attack distance between the 3-OH of the primer and the phosphorus in complexes containing Be2+ instead of Mg2+ at site A was above 4.5 A. Our findings provide a detailed description of the mechanism of DNA polymerization and should be helpful to understand the molecular basis of DNA replication fidelity.

Complex of repair DNA polymerase β with autonomous 3′→5′ exonuclease shows increased accuracy of DNA synthesis

The complexes of repair DNA polymerase beta with 3'-exonuclease and some other proteins were isolated from the chromatin of hepatocytes of normal rats for the first time. Biopolymers were extracted from the chromatin by the solution of NaCl and Triton X-100. The extract was fractionated by gel-filtration on Sephacryl S-300 columns successively in low and high ionic strength solutions, on hydroxyapatite, and on Sephadex G-100 columns. The complexes have molecular weights of 100 and 300 kDa. They dissociate to DNA polymerase and exonuclease in the course of chromatography on a DNA-cellulose column or after gel-filtration in the presence of 1 M NaCl. The co-purification of the polymerase and exonuclease is reconstituted in 0.1 M NaCl. The fidelity of monomeric and composite DNA polymerase beta was measured using phage phiX174 amber 3 as a primer/template. The products of the synthesis were transfected into Escherichia coli spheroplasts, and the frequency of reverse mutations was determined. The complex of DNA polymerase beta with 3'-exonuclease was shown to be 30 times more accurate than the monomeric polymerase, which can decrease the probability of repair mutagenesis and carcinogenesis.

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.

Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5′ to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3′ hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.

Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly

BackgroundUracil-DNA glycosylases (UDGs) catalyze excision of uracil from DNA. Vaccinia virus, which is the prototype of poxviruses, encodes a UDG (vvUDG) that is significantly different from the UDGs of other organisms in primary, secondary and tertiary structure and characteristic motifs. It adopted a novel catalysis-independent role in DNA replication that involves interaction with a viral protein, A20, to form the processivity factor. UDG:A20 association is essential for assembling of the processive DNA polymerase complex. The structure of the protein must have provisions for such interactions with A20. This paper provides the first glimpse into the structure of a poxvirus UDG.ResultsResults of dynamic light scattering experiments and native size exclusion chromatography showed that vvUDG is a dimer in solution. The dimeric assembly is also maintained in two crystal forms. The core of vvUDG is reasonably well conserved but the structure contains one additional β-sheet at each terminus. A glycerol molecule is found in the active site of the enzyme in both crystal forms. Interaction of this glycerol molecule with the protein possibly mimics the enzyme-substrate (uracil) interactions.ConclusionThe crystal structures reveal several distinctive features of vvUDG. The new structural features may have evolved for adopting novel functions in the replication machinery of poxviruses. The mode of interaction between the subunits in the dimers suggests a possible model for binding to its partner and the nature of the processivity factor in the polymerase complex.

FASTDXL: A Generalized Screen to Trap Disulfide-Stabilized Complexes for Use in Structural Studies

Structural studies of macromolecular complexes have produced extraordinary insights into a wide variety of biological processes. Unfortunately, as structural biologists pursue larger and more challenging assemblies, weakly stable and/or nonspecific interactions can become significant roadblocks to structure determination. We have developed a rapid and effective pool-based screen, termed FASTDXL (focused array screening technique for disulfide X-linking), to produce and identify disulfide-stabilized protein-nucleic acid assemblies. A significant strength of FASTDXL is that it can take advantage of prior structural knowledge about molecular interactions, but does not necessarily rely upon it. A detailed application of the approach to the difficult problem of trapping a bacterial primase-ssDNA complex is described, validating the method as a route toward obtaining diffracting crystals suitable for structure determination.

A naturally occurring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity

The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDDeltaprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix alphaH. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix-turn-helix (HTH) motif.

A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol

Thymine glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-A crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson-Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5' template guanine. The position of the displaced 5' template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.

Assignment of paramagnetic 15N-HSQC spectra by heteronuclear exchange spectroscopy

Paramagnetic metal ions in proteins provide a rich source of structural information, but the resonance assignments required to extract the information can be challenging. Here we demonstrate that paramagnetically shifted (15)N-HSQC cross-peaks can be assigned using N(Z)-exchange spectroscopy under conditions in which the paramagnetic form of the protein is in dynamic equilibrium with its diamagnetic form. Even slow exchange of specifically bound metal ions may be detected within the long lifetime of (15)N longitudinal magnetization of large proteins at high magnetic fields. Alternatively, the exchange can be accelerated using an excess of metal ions. In the resulting exchange spectra, paramagnetic (15)N resonances become visible for residues that are not directly observed in a conventional (15)N-HSQC spectrum due to paramagnetic (1)H(N) broadening. The experiments are illustrated by the 30 kDa lanthanide-binding epsilon186/theta complex of DNA polymerase III in the presence of sub-stoichiometric amounts of Dy(3+) or a mixture of Dy(3+) and La(3+).

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair.

BackgroundSeveral distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.ResultsA deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC) or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD), but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC.ConclusionOur experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC), most likely through HR, and suggest that NHEJ via Ku serves as a backup DNA repair system. The cell biological experiments show that SbcC functions in close proximity to the replication machinery, suggesting that SbcC may act on stalled or collapsed replication forks. Our results show that different patterns of localization exist for DNA repair proteins, and that the B. subtilis SMC proteins RecN and SbcC play distinct roles in the repair of DNA damage.

Involvement of 29 DNA polymerase thumb subdomain in the proper coordination of synthesis and degradation during DNA replication

φ29 DNA polymerase achieves a functional coupling between its 3′–5′ exonuclease and polymerization activities by means of important contacts with the DNA at both active sites. The placement and orientation of residues Lys538, Lys555, Lys557, Gln560, Thr571, Thr573 and Lys575 in a modelled φ29 DNA polymerase–DNA complex suggest a DNA-binding role. In addition, crystal structure of φ29 DNA polymerase–oligo (dT)5 complex showed Leu567, placed at the tip of the thumb subdomain, lying between the two 3′-terminal bases at the exonuclease site. Single replacement of these φ29 DNA polymerase residues by alanine was made, and mutant derivatives were overproduced and purified to homogeneity. The results obtained in the assay of their synthetic and degradative activities, as well as their coordination, allow us to propose: (1) a primer-terminus stabilization role at the polymerase active site for residues Lys538, Thr573 and Lys575, (2) a primer-terminus stabilization role at the exonuclease active site for residues Leu567 and Lys555 and (3) a primer-terminus binding role in both editing and polymerization modes for residue Gln560. The results presented here lead us to propose φ29 DNA polymerase thumb as the main subdomain responsible for the coordination of polymerization and exonuclease activities.

Human Cytomegalovirus (HCMV) DNA Polymerase Processivity Factor ppUL44 Dimerizes in the Cytosol before Translocation to the Nucleus

Replication of the human cytomegalovirus genome takes place in the nuclei of infected cells and is mediated by a viral-encoded DNA polymerase complex formed by the catalytic subunit pUL54 and the processivity factor ppUL44. Although it has recently been shown that the dimerization ability of recombinant pUL44 appears to be crucial for effective DNA binding in vitro, whether ppUL44 can dimerize or not in a cellular context is unknown. Here, we show, by using co-immunoprecipitation and dual-color live imaging approaches on cells expressing fluorescent and differently tagged ppUL44 fusion proteins, that ppUL44 dimerizes in the cytoplasm via its N-terminal domain, before translocating to the nucleus. Furthermore, we show that nuclear translocation of differently tagged ppUL44 heterodimers can occur even when one subunit carries a nonfunctional nuclear localization signal. Importantly, the latter cotransfection assay represents a system to test small-molecule inhibitors for their ability to impair ppUL44 dimerization.

Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: purification and crystallization of the DNA polymerase and PCNA monomer mutant complex fromPyrococcus furiosus

Replicative DNA polymerase interacts with processivity factors, the beta-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 A resolution using synchrotron radiation. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 A.

Using 2-Aminopurine Fluorescence To Detect Bacteriophage T4 DNA Polymerase−DNA Complexes That Are Important for Primer Extension and Proofreading Reactions

The fluorescence of the base analogue 2-aminopurine (2AP) was used to probe bacteriophage T4 DNA polymerase-induced conformational changes in the template strand produced during the nucleotide incorporation and proofreading reactions. 2AP fluorescence in DNA is quenched by 2AP interactions with neighboring bases, but T4 DNA polymerase binding to DNA substrates labeled with 2AP in the templating position produces large increases in fluorescence intensity. Fluorescence lifetime studies were performed to characterize the fluorescent complexes. Three fluorescence lifetime components were observed for unbound DNA substrates as reported previously, but T4 DNA polymerase binding modulated the amplitudes of these components and created a new, highly fluorescent 10.5 ns component. Experimental evidence for correlation of fluorescence lifetimes with functionally distinct complexes was obtained by forming complexes under different reaction conditions. T4 DNA polymerase complexes were formed with DNA substrates with matched and mismatched primer ends and with A+T- or G+C-rich primer-terminal regions. dTTP was added to binary complexes to form ternary DNA polymerase-DNA-nucleotide complexes. The effect of temperature on complex formation was studied, and complexes were formed with proofreading-defective T4 DNA polymerases. Complexes characterized by the 10.5 ns lifetime were demonstrated to be formed at the crossroads of the primer-extension and proofreading pathways.