The mechanism of nucleotide incorporation by human DNA polymerase eta differs from that of the yeast enzyme

DNA polymerase eta (Poleta) catalyzes the efficient and accurate synthesis of DNA opposite cyclobutane pyrimidine dimers, and inactivation of Poleta in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Pre-steady-state kinetic studies of yeast Poleta have indicated that the low level of fidelity of this enzyme results from a poorly discriminating induced-fit mechanism. Here we examine the mechanistic basis of the low level of fidelity of human Poleta. Because the human and yeast enzymes behave similarly under steady-state conditions, we expected these enzymes to utilize similar mechanisms of nucleotide incorporation. Surprisingly, however, we find that human Poleta differs from the yeast enzyme in several important respects. The human enzyme has a 50-fold-faster rate of nucleotide incorporation than the yeast enzyme but binds the nucleotide with an approximately 50-fold-lower level of affinity. This lower level of binding affinity might provide a means of regulation whereby the human enzyme remains relatively inactive except when the cellular deoxynucleoside triphosphate concentrations are high, as may occur during DNA damage, thereby avoiding the mutagenic consequences arising from the inadvertent action of this enzyme during normal DNA replication.     

Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase kappa

The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Poleta, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polkappa, which differs from Poleta in having a higher fidelity and which, unlike Poleta, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Poleta. We find that an isosteric thymidine analog is replicated with low efficiency by Polkappa, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Poleta and Polkappa rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.     

Interaction with PCNA is essential for yeast DNA polymerase eta function.

In both yeast and humans, DNA polymerase (Pol) eta functions in error-free replication of ultraviolet-damaged DNA, and Poleta promotes replication through many other DNA lesions as well. Here, we present evidence for the physical and functional interaction of yeast Poleta with proliferating cell nuclear antigen (PCNA) and show that the interaction with PCNA is essential for the in vivo function of Poleta. Poleta is highly inefficient at inserting a nucleotide opposite an abasic site, but interaction with PCNA greatly stimulates its ability for nucleotide incorporation opposite this lesion. Thus, in addition to having a pivotal role in the targeting of Poleta to the replication machinery stalled at DNA lesions, interaction with PCNA would promote the bypass of certain DNA lesions.     

Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta

Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase eta (Poleta) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Poleta are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Poleta; in this regard, Poleta differs strikingly from classical high-fidelity DNA polymerases.     

Subtle but variable conformational rearrangements in the replication cycle of Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) may accommodate lesion bypass

The possible conformational changes of DNA polymerase IV (Dpo4) before and after the nucleotidyl-transfer reaction are investigated at the atomic level by dynamics simulations to gain insight into the mechanism of low-fidelity polymerases and identify slow and possibly critical steps. The absence of significant conformational changes in Dpo4 before chemistry when the incoming nucleotide is removed supports the notion that the "induced-fit" mechanism employed to interpret fidelity in some replicative and repair DNA polymerases does not exist in Dpo4. However, significant correlated movements in the little finger and finger domains, as well as DNA sliding and subtle catalytic-residue rearrangements, occur after the chemical reaction when both active-site metal ions are released. Subsequently, Dpo4's little finger grips the DNA through two arginine residues and pushes it forward. These metal ion correlated movements may define subtle, and possibly characteristic, conformational adjustments that operate in some Y-family polymerase members in lieu of the prominent subdomain motions required for catalytic cycling in other DNA polymerases like polymerase beta. Such subtle changes do not easily provide a tight fit for correct incoming substrates as in higher-fidelity polymerases, but introduce in low-fidelity polymerases different fidelity checks as well as the variable conformational-mobility potential required to bypass different lesions.     

Role of DNA polymerase zeta in the bypass of a (6-4) TT photoproduct

UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA polymerase eta (Poleta), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Poleta result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Here, we test Poleta for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than a cis-syn TT dimer. Opposite the 3' T of a (6-4) TT photoproduct, both yeast and human Poleta preferentially insert a G residue, but they are unable to extend from the inserted nucleotide. DNA Polzeta, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3' T of the (6-4) TT lesion by Poleta, and Polzeta inserts the correct nucleotide A opposite the 5' T of the lesion. Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Poleta and Polzeta, wherein Poleta inserts a nucleotide opposite the 3' T of the lesion and Polzeta extends from it. These biochemical observations are in concert with genetic studies in yeast indicating that mutations occur predominantly at the 3' T of the (6-4) TT photoproduct and that these mutations frequently exhibit a 3' T-->C change that would result from the insertion of a G opposite the 3' T of the (6-4) TT lesion.     

Mismatch extension ability of yeast and human DNA polymerase eta

DNA polymerase eta (Poleta) functions in error-free replication of UV-damaged DNA, and in vitro it efficiently bypasses a cis-syn T-T dimer by incorporating two adenines opposite the lesion. Steady state kinetic studies have shown that both yeast and human Poleta are low-fidelity enzymes, and they misincorporate nucleotides with a frequency of 10(-2)-10(-3) on both undamaged and T-T dimer-containing DNA templates. To better understand the role of Poleta in error-free translesion DNA synthesis, here we examine the ability of Poleta to extend from base mismatches. We find that both yeast and human Poleta extend from mismatched base pairs with a frequency of approximately 10(-3) relative to matched base pairs. In the absence of efficient extension of mismatched primer termini, the ensuing dissociation of Poleta from DNA may favor the excision of mismatched nucleotides by a proofreading exonuclease. Thus, we expect DNA synthesis by Poleta to be more accurate than that predicted from the fidelity of nucleotide incorporation alone.     

Identification of a strand-related bias in the PCNA-mediated bypass of spontaneous lesions by yeast Poleta

Translesion synthesis (TLS) DNA polymerases are specialized to bypass lesions that block replicative polymerases and prevent complete genome duplication. Current TLS models hypothesize that PCNA, the polymerase processivity clamp, is important for regulating the access and loading of the low fidelity TLS polymerases onto DNA in response to replication-blocking lesions. PCNA binds to the C-terminus of yeast Poleta, for example, and this interaction is required for cell survival after UV irradiation. Previously, we identified two spontaneous, Polzeta-dependent "complex" mutation hotspots using the lys2DeltaA746 frameshift reversion assay in repair-compromised cells. In the current study we observed an accumulation of Polzeta-dependent complex frameshifts at a third hotspot in Poleta-deficient cells. Interestingly, the sequence of this third hotspot is the reverse complement of the two hotspots previously identified, suggesting that the utilization of Polzeta and Poleta may be related to the position of the relevant lesion on either the leading- or lagging-strand template. Using the lys2DeltaA746 assay system, we investigated changes in the accumulation of complex events at hotspots when the direction of replication was reversed in repair-compromised cells with either wildtype Poleta, a deletion of Poleta, or a mutant of Poleta that cannot interact with PCNA. Our results suggest that there is a polymerase hierarchy between Poleta and Polzeta in the bypass of certain lesions and that the interaction of Poleta with PCNA is needed for some, but not all, spontaneous lesion bypass.     

Mismatched base-pair simulations for ASFV Pol X/DNA complexes help interpret frequent G*G misincorporation

DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G*G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts G*G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C*C, A*G, and G*G (anti) and A*G and G*G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G*G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G*C). The base pairing in the G*G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A*G and G*G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C*C and the A*G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A*G anti to worst for the C*C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol beta, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol beta active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.     

A reexamination of the nucleotide incorporation fidelity of DNA polymerases

Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.     

Two-step error-prone bypass of the (+)- and (-)-trans-anti-BPDE-N2-dG adducts by human DNA polymerases eta and kappa

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase eta (Poleta) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Poleta predominantly inserted an A opposite a template (+)- and (-)-trans-anti-BPDE-N2-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Poleta. Error-prone nucleotide insertion by human Poleta was more efficient opposite the (+)-trans-anti-BPDE-N2-dG adduct than opposite the (-)-trans-anti-BPDE-N2-dG. However, translesion synthesis by human Poleta largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Poleta from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N2-dG adducts, the nucleotide opposite the lesion, and the sequence context 5' to the lesion. By combining the nucleotide insertion activity of human Poleta and the extension synthesis activity of human Polkappa, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts.     

In silico evidence for DNA polymerase-beta's substrate-induced conformational change

Structural information for mammalian DNA pol-beta combined with molecular and essential dynamics studies have provided atomistically detailed views of functionally important conformational rearrangements that occur during DNA repair and replication. This conformational closing before the chemical reaction is explored in this work as a function of the bound substrate. Anchors for our study are available in crystallographic structures of the DNA pol-beta in "open" (polymerase bound to gapped DNA) and "closed" (polymerase bound to gapped DNA and substrate, dCTP) forms; these different states have long been used to deduce that a large-scale conformational change may help the polymerase choose the correct nucleotide, and hence monitor DNA synthesis fidelity, through an "induced-fit" mechanism. However, the existence of open states with bound substrate and closed states without substrates suggest that substrate-induced conformational closing may be more subtle. Our dynamics simulations of two pol-beta/DNA systems (with/without substrates at the active site) reveal the large-scale closing motions of the thumb and 8-kDa subdomains in the presence of the correct substrate--leading to nearly perfect rearrangement of residues in the active site for the subsequent chemical step of nucleotidyl transfer--in contrast to an opening trend when the substrate is absent, leading to complete disassembly of the active site residues. These studies thus provide in silico evidence for the substrate-induced conformational rearrangements, as widely assumed based on a variety of crystallographic open and closed complexes. Further details gleaned from essential dynamics analyses clarify functionally relevant global motions of the polymerase-beta/DNA complex as required to prepare the system for the chemical reaction of nucleotide extension.     

Mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase IV

The kinetic mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is resolved by pre-steady-state kinetic analysis of single-nucleotide (dTTP) incorporation into a DNA 21/41-mer. Like replicative DNA polymerases, Dpo4 utilizes an "induced-fit" mechanism to select correct incoming nucleotides. The affinity of DNA and a matched incoming nucleotide for Dpo4 was measured to be 10.6 nM and 230 microM, respectively. Dpo4 binds DNA with an affinity similar to that of replicative polymerases due to the presence of an atypical little finger domain and a highly charged tether that links this novel domain to its small thumb domain. On the basis of the elemental effect between the incorporations of dTTP and its thio analogue S(p)-dTTPalphaS, the incorporation of a correct incoming nucleotide by Dpo4 was shown to be limited by the protein conformational change step preceding the chemistry step. In contrast, the chemistry step limited the incorporation of an incorrect nucleotide. The measured dissociation rates of the enzyme.DNA binary complex (0.02-0.07 s(-1)), the enzyme.DNA.dNTP ternary complex (0.41 s(-1)), and the ternary complex after the protein conformational change (0.004 s(-1)) are significantly different and support the existence of a bona fide protein conformational change step. The rate-limiting protein conformational change was further substantiated by the observation of different reaction amplitudes between pulse-quench and pulse-chase experiments. Additionally, the processivity of Dpo4 was calculated to be 16 at 37 degrees C from analysis of a processive polymerization experiment. The structural basis for both the protein conformational change and the low processivity of Dpo4 was discussed.     

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.     

Stimulation of DNA Synthesis Activity of Human DNA Polymerase κ by PCNA

Humans have three DNA polymerases, Poleta, Polkappa, and Poliota, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polkappa (hPolkappa) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolkappa. The processivity of hPolkappa, however, is not significantly increased in the presence of these protein factors. The efficiency (V(max)/K(m)) of correct nucleotide incorporation by hPolkappa is enhanced approximately 50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K(m) for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased approximately 40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolkappa would bypass an abasic site by inserting a nucleotide opposite the site.     

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.     

Replication past O(6)-methylguanine by yeast and human DNA polymerase eta

O(6)-Methylguanine (m6G) is formed by the action of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase eta (Poleta) in the replicative bypass of m6G lesions in DNA. The formation of MNNG-induced mutations is almost abolished in the rad30Delta pol32Delta double mutant of yeast, which lacks the RAD30 gene that encodes Poleta and the Pol32 subunit of DNA polymerase delta (Poldelta). Although Poldelta can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Poleta is much more efficient in replicating through m6G than Poldelta. Both Poleta and Poldelta insert a C or a T residue opposite from m6G; Poleta, however, is more accurate, as it inserts a C about twice as frequently as Poldelta. Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA. Inactivation of Poleta could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy.     

Mechanistic consequences of temperature on DNA polymerization catalyzed by a Y-family DNA polymerase

Our previous publication shows that Sulfolobus solfataricus Dpo4 utilizes an ‘induced-fit’ mechanism to select correct incoming nucleotides at 37°C. Here, we provide a comprehensive report elucidating the kinetic mechanism of a DNA polymerase at a reaction temperature higher than 37°C in an attempt to determine the effect of temperature on enzyme fidelity and mechanism. The fidelity of Dpo4 did not change considerably with a 30°C increase in reaction temperature, suggesting that the fidelity of Dpo4 at 80°C is similar to that determined here at 56°C. Amazingly, the incorporation rate for correct nucleotides increased by 18 900-fold from 2°C to 56°C, similar in magnitude to that observed for incorrect nucleotides, thus not perturbing fidelity. Three independent lines of kinetic evidence indicate that a protein conformational change limits correct nucleotide incorporations at 56°C. Furthermore, the activation energy for the incorporation of a correct nucleotide was determined to be 32.9 kcal/mol, a value considerably larger than those values estimated for a rate-limiting chemistry step, providing a fourth line of evidence to further substantiate this conclusion. These results herein provide evidence that Dpo4 utilizes the ‘induced-fit’ mechanism to select a correct nucleotide at all temperatures.     

Efficiency of correct nucleotide insertion governs DNA polymerase fidelity

DNA polymerase fidelity or specificity expresses the ability of a polymerase to select a correct nucleoside triphosphate (dNTP) from a pool of structurally similar molecules. Fidelity is quantified from the ratio of specificity constants (catalytic efficiencies) for alternate substrates (i.e. correct and incorrect dNTPs). An analysis of the efficiency of dNTP (correct and incorrect) insertion for a low fidelity mutant of DNA polymerase beta (R283A) and exonuclease-deficient DNA polymerases from five families derived from a variety of biological sources reveals that a strong correlation exists between the ability to synthesize DNA and the probability that the polymerase will make a mistake (i.e. base substitution error). Unexpectedly, this analysis indicates that the difference between low and high fidelity DNA polymerases is related to the efficiency of correct, but not incorrect, nucleotide insertion. In contrast to the loss of fidelity observed with the catalytically inefficient R283A mutant, the fidelity of another inefficient mutant of DNA polymerase beta (G274P) is not altered. Thus, although all natural low fidelity DNA polymerases are inefficient, not every inefficient DNA polymerase has low fidelity. Low fidelity polymerases appear to be an evolutionary solution to how to replicate damaged DNA or DNA repair intermediates without burdening the genome with excessive polymerase-initiated errors.     

Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis.