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Recently, we synthesized the first individual β,γ-CHX-dGTP diastereomers [(R)- or (S)-CHX, where X is F or Cl] and determined their structures in ternary complexes with DNA polymerase β (pol β). We now report stereospecificity by pol β on the mixed β,γ-CHX diastereomer pairs using nuclear magnetic resonance and on the separate diastereomers using transient kinetics. For both the F and Cl diastereomers, the R isomer is favored over the S isomer for G·C correct incorporation, with stereospecificities [(k(pol)/K(d))(R)/(k(pol)/K(d))(S)] of 3.8 and 6.3, respectively, and also for G·T misincorporation, with stereospecificities of 11 and 7.8, respectively. Stereopreference for the (R)-CHF-dGTP diastereomer was abolished for k(pol) but not K(d) with mutant pol β (R183A). These compounds constitute a new class of stereochemical probes for active site interactions involving halogen atoms. As Arg183 is unique in family X pols, the design of CXY deoxyribonucleotide analogues to enhance interaction is a possible strategy for inhibiting BER selectively in cancer cells.

Human 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme involved in removing 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic DNA lesion generated by oxidative stress. The removal of 8-oxoG by OGG1 is affected by the local DNA sequence, and this feature most likely contributes to observed mutational hot spots in genomic DNA. To elucidate the influence of local DNA sequence on 8-oxoG excision activity of OGG1, we conducted steady-state, pre-steady-state, and single turnover kinetic evaluation of OGG1 in alternate DNA sequence contexts. The sequence context effect was studied for a mutational hot spot at a CpG dinucleotide. Altering either the global DNA sequence or the 5′-flanking unmodified base pair failed to influence the excision of 8-oxoG. Methylation of the cytosine 5′ to 8-oxoG also did not affect 8-oxoG excision. In contrast, a 5′-neighboring mismatch strongly decreased the rate of 8-oxoG base removal. Substituting the 5′-C in the CpG dinucleotide with T, A, or tetrahydrofuran (i.e. T:G, A:G, and tetrahydrofuran:G mispairs) resulted in a 10-, 13-, and 4-fold decrease in the rate constant for 8-oxoG excision, respectively. A greater loss in activity was observed when T:C or A:C was positioned 5′ of 8-oxoG (59- and 108-fold, respectively). These results indicate that neighboring structural abnormalities 5′ to 8-oxoG deter its repair thereby enhancing its mutagenic potential.

DNA polymerase (pol) β is a member of the X-family of DNA polymerases and plays an important role in base excision repair that cleanses the genome of simple base lesions.[1] It has been the subject of extensive studies examining its key roles in repair[2] and cancer.[3] In an effort to identify features of the pol β active site that modulate nucleotide binding, we have made modifications in the triphosphate moiety and studied the effect on enzyme binding, specificity, and chemistry.[4] Replacement of the Pα-O-Pβ bridging oxygen with a carbon atom prevents catalysis,[4b] whereas modification of the Pβ-O-Pγ bridging oxygen alters leaving group properties.[4a] The introduction of these substituents may also enable entirely new bonding (or repulsive) active site interactions, not present with the natural nucleoside triphosphate, and thus could inform mechanistic insight as well as inhibitor design seeking to exploit pol β as a drug target.[4a] Previously we studied several α,β-methylene-dNTP analogues as probes for the capture of pre-insertion substrate complexes with pol β and found that α,β-CF2-analogues have an apparent equilibrium dissociation constant (Kd) that is two orders of magnitude greater than the corresponding α,β-CH2-analogues and the β,γ-dGTP-CF2-analogue.[4a, 4b] To further explore the molecular basis of pol β nucleotide binding, the dimethylated (α,β-C(Me)2-, (5)) and the monofluorinated (R/S)-(α,β-CHF-, (6a/b)) dATP analogues have been synthesized and their Kd values determined. In addition, the X-ray crystallographic structures of ternary substrate complexes of pol β and DNA primer template formed from α,β-CF2-dATP[4b] (7) and 6a/b have been solved. Interestingly, only one diastereomer of 6 is found in the active site (Figure 1). Figure 1 X-ray crystal structure of the DNA pol β active site containing (S)-α,β-CHF-dATP. The solvent-excluded pol β active site surface is colored by atom (carbon, gray; nitrogen, light blue; oxygen, pink; magnesium, dark blue ... 5 and 6a/b were prepared by reaction of 2′-deoxyadenosine 5′-tosylate with the tris(tetrabutylammonium) salt of the corresponding bisphosphonic acid followed by enzymatic phosphorylation of the resulting dA 5′-bisphosphonate[4b] (Scheme 1). In a previously reported synthesis of 6a/b in which (R/S)-α,β-CHF-dADP (4a/b) was phosphorylated with p-nitrobenzyl phosphormorpholidate,[5] an approximately 1:1 mixture of α,β-CHF-dATP diastereomers was observed in the 19F NMR spectrum. Similarly, phosphorylation of 4a/b using nucleoside diphosphate kinase and a catalytic amount of ATP generates both diastereomers as observed in the 19F and 31P (Pα) NMR spectra. Although precise quantification is not possible due to signal overlap, by 19F NMR analysis the isomers are in roughly similar abundance, indicating that the enzymatic phosphorylation did not significantly enrich a particular isomer. Scheme 1 Synthesis of α,β-CXY-dATP analogues. As expected, 5 and 6a/b are not pol β substrates (SI, Figure S25) and inhibit gap-filling DNA synthesis by pol β in a concentration dependent manner (SI, Figures S26 and S28). The apparent equilibrium binding constant for 6a/b is approximately 10 μM, similar to that of the natural substrate and significantly lower than that of the α,β-CF2-dATP analogue (Figure 2).[4b] The α,β-CH2-dATP analogue binds with about a 10-fold higher affinity[4b, 6] than 6a/b consistent with a correlation between the basicity of the phosphonate moiety and the strength of inhibitor binding.[4b] However, α,β-C(Me)2-dATP (5) which has two electron-donating substituents on the α,β-methylene, has a Kd of approximately 1100 μM and does not conform to this trend. Figure 2 DNA pol β dissociation constants of dATP and α,β-methylene analogues. The data for dATP, α,β-CH2-dATP, and α,β-CF2-dATP were previously published.[4b, 9] The Ki (i.e., Kd) values were determined ... The crystallographic structures of ternary complexes of pol β with incoming α,β-CF2-dATP (7) (PDB ID 3TFR) or α,β-CHF-dATP (6) (PDB ID 3TFS) opposite a templating thymidine, resolved at 2.00 A (SI Table S1) were next determined. All corresponding atoms in the active site of these structures superimpose well with previously determined ternary complex structures of pol β where the reaction was trapped by deletion of the nucleophilic 3′-OH on the primer terminus or by using a nitrogen in place of the α,β-bridging oxygen (Figure 3A).[8] Thus, α,β-CHF-dATP and 7 are well accommodated in the polymerase active site as has been shown previously for α,β-CF2-dTTP.[4b] Figure 3 A) The ternary complex crystallographic structures of pol β with an incoming α,β-CF2-dATP (7) (gray carbons) and α,β-NH-dUTP (light blue carbons; PDB ID 2FMS)[8] were superimposed using all 326 Cα (rmsd ... Molecular docking of 5 and 6 with pol β-DNA binary complex using Autodock Vina[7] suggests that the unexpectedly low binding affinity of 5 can be attributed to an unfavorable steric interaction, specifically a clash of the methyl groups of 5 with a structural water bound to Asp276 (PDB ID 3TFS water 3). When docked in the absence of this structural water, the lowest energy conformations for both 5 and 6 overlap well with the coordinates of the 6 found in the crystal structure, whose conformation is congruent with those of DNA pol β-bound α,β-CF2-dTTP[4b] and α,β-NH-dUTP.[8] However, when docking using a macromolecule file prepared to include the water bound to Asp276, 5 is unable to achieve the experimentally observed conformation while the geometry of 6 is unperturbed (SI, Figure S27). Incubation of crystals of a binary DNA complex of pol β with the α,β-CHF-dATP diastereomer mixture (6a/b) resulted in a complex in which only the (S)-CHF-stereoisomer (6b) could be found by X-ray analysis (Figures 1 and ​and3B).3B). Modeling (R)-α,β-CFH-dATP (6a) into the electron density failed to account for the observed density (Figure 3C). The close overlap of the CF2- and the (S)-CHF- conformations indicates that exclusion of 6a analogue is not the result of an unattainable binding conformation or destabilizing steric interaction (Figure 3B). As we reasoned previously,[4d, 4e] stabilization of as little as ~1 kcal/mol could be sufficient to generate the selectivity observed in the crystal structures. In an earlier study, we reported a series of stereospecifically formed pol β-DNA-dGTP ternary complexes that provided evidence for a stabilizing polar interaction between the active site Arg183 and the chiral phosphonate C-F in CHF, CMeF, and CClF dGTP-β,γ analogues.[4d, 4e] Unlike the β,γ-CXF-dGTP-DNA-pol β ternary complexes, in this structure no direct stabilizing interactions with active site residues can be identified. The structure does, however, reveal that the (S)-isomer fluorine is within 2.8 A (B-factor: 15.4 A2) of an oxygen in a structural water molecule, present in both structures (for CF2 distance = 2.9 A, B-factor: 15.1 A2), that is within hydrogen bonding distance of several key atoms (a carboxylate anion oxygen and carboxyamide nitrogen of Asp276, phosphoryl oxygen of Pβ; Figure 1). Observing no similar interactions with the pro-(R)-fluorine in the CF2-structure, one might consider a weak interaction of the (S)-fluorine in 6b with structural water as the source of its stabilization in the complex relative to 6a, however other factors might be invoked[10] and further work will be necessary to clarify the origin of the observed stereospecificity. In summary, our results show the influence of a structural water bound to Asp276 on the nucleotide binding properties of DNA pol β. Kd determinations for a series of α,β-bridging substituted dATP analogues display a correlation with the basicity of the bisphosphonate moiety and the binding affinity to pol β. This trend, however, does not extend to the α,β-C(Me)2-dATP analogue which displays a larger than expected Kd attributed to an unfavorable steric interaction of the methyl substituents with the structural water bound to Asp276. The X-ray crystal structure of α,β-CHF-dATP in complex with pol β and DNA contains only one diastereomer despite the presence of both diastereomers in the crystallization mixture. Comparison of this structure with that of α,β-CF2-dATP raises the possibility of a non-covalent weak interaction between the ligand and the active site structural water proximal to Asp276. Our structural observations may be worth taking into account in PDB studies[11] searching for fluorine/protein interactions.

Human immunodeficiency virus type 1 reverse transcriptase has subunits of 66 and 51 kDa (p66 and p51, respectively). Structural studies indicate that each subunit consists of common subdomains. The polymerase domain of p66 forms a nucleic acid binding cleft, and, by analogy with a right hand, the subdomains are referred to as fingers, palm, and thumb (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Residues 257-266 correspond to a highly conserved region of primary structure among retroviral pol genes. Crystallographic evidence indicates that these residues are in the thumb subdomain and form part of an alpha-helix (alpha H), which interacts with DNA (Jacobo-Molina, A., Ding, J., Nanni, R. G., Clark, A. D., Jr., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H., and Arnold, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6320-6324). To define the role of this region during catalytic cycling, we performed systematic site-directed mutagenesis from position 253 through position 271 by changing each residue, one by one, to alanine. Each mutant protein was expressed and purified, and their substrate-specific activities were surveyed. The results are consistent with alpha H (residues 255-268) of p66 interacting with the template and/or primer strand. The core of alpha H appears to play an important role in template-primer binding (residues Gln-258, Gly-262, and Trp-266), and in protein-protein interactions (residues Val-261 and Leu-264). The periodicity of the effects observed suggest that a segment of one face of alpha H interacts with the template-primer. The lower fidelity observed with alanine mutants of Gly-262 and Trp-266 correlated with an over 200-fold increase in the dissociation rate constant for template-primer relative to wild type enzyme and suggests that enzyme-DNA interactions in the template-primer stem are important fidelity determinants.

Purified mammalian DNA polymerase beta (beta-pol) fills short gaps of up to 6 nucleotides by a processive mechanism, and this gap-filling activity requires a PO4 group on the 5'-side of the gap (Singhal, R. K., and Wilson, S. H. (1993) J. Biol. Chem. 268, 15906-15911). To assess details of bimolecular binding between beta-pol and a 5-nucleotide (nt) gapped radiolabeled heteropolymeric DNA substrate, beta-pol.DNA complexes were formed, photochemically cross-linked using UV light, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A 39-nt template was annealed with two 17-mer oligonucleotides, generating a 5-nt gap. The results indicate that beta-pol binds to both the template and primer strands, and binding is strongly enhanced by a 5'-PO4 on the downstream oligonucleotide, even though little cross-linking is observed to this oligonucleotide. The results suggest that beta-pol recognizes the 5'-side of a long single-stranded gap in DNA, provided it contains a 5'-PO4. Additional beta-pol.DNA binding measurements were performed using a competition assay to assess the ability of heteropolymeric DNA to inhibit synthesis on a homopolymeric template-primer system. The results indicate that in addition to the 5'-PO4, the length of the single-stranded template nucleic acid adjacent to the 5'-PO4 is also important for tight binding. Proteolysis of the cross-linked beta-pol.DNA complex with trypsin resulted in a single radiolabeled tryptic product corresponding to nucleic acid cross-linked to the 8-kDa domain. The results demonstrate that the role of the 8-kDa domain is to direct beta-pol binding to the phosphorylated 5'-position in gapped DNA substrates.

The core promoter of the human DNA beta-polymerase (beta-pol) gene is regulated by proteins binding at 3 GC boxes and the single activating transcription factor/cAMP response element (ATF/CRE) centered at -45; the central 8 residues of this ATF/CRE match the ATF/CRE consensus sequence, TGACGTCA. Previously, we purified a beta-pol promoter ATF/CRE-binding protein (named palindrome-binding protein or PBP) from bovine testes and found that this protein is a beta-pol promoter transcriptional activator in vitro using a HeLa nuclear extract transcription system (Widen, S. G., and Wilson, S. H. (1991) Biochemistry 30, 6296-6305). In this study, we determined the mechanism of in vitro transcriptional activation by this purified PBP. We used a PBP-depleted HeLa nuclear extract transcription system with an artificial promoter containing a solitary activator element corresponding to the entire 22-nucleotide beta-pol promoter ATF/CRE-binding site. Kinetic analyses of the 180-nucleotide run-off product formation indicated that stimulation of transcriptional activity by PBP was due entirely to an increase in the rate constant for promoter clearance. Thus, under our conditions, the purified PBP had no effect on the rate of closed preinitiation complex formation or for the closed complex to open complex transition. Instead, the rate of productive initiation leading to the 180-nucleotide transcript was stimulated by PBP. We found that the rate of closed preinitiation complex formation was not in rapid equilibrium with promoter and RNA polymerase II, in contrast to the model with prokaryotic RNA polymerase transcription. The results also indicated that PBP binding to the ATF/CRE is required for the stimulation of promoter clearance. These studies define the kinetic mechanism of a purified ATF/CRE-binding protein in stimulation of the in vitro transcription of a designed mammalian promoter.

Oxidation of genomic DNA forms the guanine lesion 7,8-dihydro-8-oxoguanine (8-oxoG). When in the template base position during DNA synthesis the 8-oxoG lesion has dual coding potential by virtue of its anti - and syn -conformations, base pairing with cytosine and adenine, respectively. This impacts mutagenesis, because insertion of adenine opposite template 8-oxoG can result in a G to T transversion. DNA polymerases vary by orders of magnitude in their preferences for mutagenic vs. error-free 8-oxoG lesion bypass. Yet, the structural basis for lesion bypass specificity is not well understood. The DNA base excision repair enzyme DNA polymerase (pol) β is presented with gap-filling synthesis opposite 8-oxoG during repair and has similar insertion efficiencies for dCTP and dATP. We report the structure of pol β in binary complex with template 8-oxoG in a base excision repair substrate. The structure reveals both the syn - and anti -conformations of template 8-oxoG in the confines of the polymerase active site, consistent with the dual coding observed kinetically for this enzyme. A ternary complex structure of pol β with the syn -8-oxoG: anti -A Hoogsteen base pair in the closed fully assembled preinsertion active site is also reported. The syn -conformation of 8-oxoG is stabilized by minor groove hydrogen bonding between the side chain of Arg283 and O8 of 8-oxoG. An adjustment in the position of the phosphodiester backbone 5′-phosphate enables 8-oxoG to adopt the syn -conformation.

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase β discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn(2+) for Mg(2+) decreased sugar discrimination for both wild-type and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A- and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2' of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (∼2.5 and 2.6 A, respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ∼2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase β utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

Virion-derived HIV-1 reverse transcriptase (RT) has subunits of molecular mass 66 and 51 kDa (p66 and p51, respectively) in an approximately 1:1 ratio. Since enzyme activity appears to depend on dimerization of these subunits, identification of critical regions of primary sequence required for proper dimerization could lead to potential targets for antiviral therapy. A central region of primary sequence contains a leucine hepta-repeat motif from leucine 282 to leucine 310 that has been suggested to be involved in dimerization [Baillon, J. G., Nashed, N. T., Kumar, A., Wilson, S. H., & Jerina, D. M. (1991) New Biol. 3, 1015-1019]. A region including this hepta-repeat was recently shown to be involved in protein-protein interactions required for dimerization [Becerra, S. P., Kumar, A., Lewis, M. S., Widen, S. G., Abbotts, J., Karawya, E. M., Hughes, S. H., Shiloach, J., & Wilson, S. H. (1991) Biochemistry 30, 11708-11719]. To investigate the role of this repeat motif in dimerization, we performed site-directed mutagenesis of these leucine residues from position 282 to position 310. Mutations were introduced into p66 and p51 RT coding sequences, and the individually purified RT subunit polypeptides were compared with wild-type polypeptides for dimerization. Physical characterization of the purified mutant peptides was conducted by circular dichroism analysis. Binding between p66 and p51 was studied by gel filtration, ultracentrifugation, and CD analysis. L289K-p66 was unable to dimerize with itself and wild-type or L289K-p51. The leucine repeat motif in the p66 subunit appears to be critical in formation of the heterodimer.(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylates (2-5A) and derivatives are noncompetitive inhibitors of primer/HIV-1 reverse transcriptase complex formation. The mechanism and specificity of this inhibitory action of 2-5A and 2-5A derivatives have been evaluated with 2-5A molecules modified in ribosyl moiety, chain length, extent of 5'-phosphorylation, and 2',5'-phosphodiester linkage. UV covalent cross-linking of preformed complexes of p66/p66 homodimer or p66/p51 heterodimer recombinant HIV-1 reverse transcriptase and the primer analog pd(T)16 allowed analysis of the initial step in HIV-1 reverse transcriptase-catalyzed DNA synthesis. Utilizing this primer binding assay, it is demonstrated that 2-5A and 2-5A derivatives inhibit the binding of pd(T)16 to HIV-1 reverse transcriptase. This inhibition is specific for the 2',5'-internucleotide linkage in that the corresponding 3',5'-adenylate derivatives do not exhibit inhibitory activity. Enhanced inhibitory properties were observed following modifications of the 2-5A molecule which result in an increase in hydrophobicity. Replacement of the D-ribosyl moiety of 2-5A with the 3'-deoxyribosyl moiety increased the inhibition of primer/HIV-1 reverse transcriptase complex formation 15-20%. 2',5'-Phosphorothioate substitution yielded the most effective inhibitors, with Ki's of 7-13 microM. In all cases, inhibition of primer/HIV-1 reverse transcriptase complex formation showed a preference for the 5'-triphosphate moiety. Nonphosphorylated derivatives were not inhibitory; 5'-monophosphate derivatives exhibited little or no inhibition. The inhibition of primer binding to HIV-1 reverse transcriptase correlated well with the inhibition of DNA-directed DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) exists predominantly as a heterodimer (p66/p51), but can also form a homodimer of p66 subunits (p66/p66). RT binds to template-primer (T/P) tightly to form the first complex in the reaction sequence poised to conduct DNA synthesis upon the addition of dNTP and Mg2+. We have made use of this property to kinetically analyze poly(rA)-(dT)n interactions with recombinant homo- and heterodimeric HIV-1 RT derived from HXB2R proviral DNA. A T/P challenge assay was used to quantitatively follow RT-T/P complex formation. The homo- and heterodimeric forms of RT bound to poly(rA)-(dT)16 in a kinetically similar fashion. There was no more than a 2-fold difference in kcat or for any T/P parameter examined: Km, Kd, kon, koff determined from a binary complex or from a complex incorporating dTMP, processivity, and stoichiometry of binding. In contrast, it was found that the T/P Km with heterodimeric RT derived from the NY5 strain was significantly greater than that determined for HXB2R enzyme, indicating that a kinetic diversity exists between RT derived from different viral strains. Since HXB2R RT binds to poly(rA)-(dT)16 tightly, Kd < 1 nM, active-site titrations are facilitated. At saturation, one T/P binds per two polypeptides, suggesting that RT binds substrate productively as a dimer and that if monomers are present they must rapidly form dimers in the presence of T/P.(ABSTRACT TRUNCATED AT 250 WORDS)

The African swine fever virus (ASFV) is a DNA virus that codes for two DNA polymerases: a B-family replicative DNA polymerase and a DNA polymerase (pol X) of unknown function that has sequence similarity to DNA polymerase β (pol β), a eukaryotic base excision repair enzyme in the X-family of polymerases1. Pol X is the smallest naturally occurring polymerase (174 residues, 20 kDa) and lacks accessory activities, such as the lyase activity associated with pol β. The small size of pol X makes this enzyme an ideal system for characterization by NMR. As reported on pages 936 and 942 of this issue of Nature Structural Biology, Maciejewski et al.2 and Showalter et al.3 have independently determined the solution structure of ASFV pol X. These structures represent the first solution structures of a full-length DNA polymerase. More importantly, the pol X structures provide valuable insights into how pol X can tightly bind DNA in the absence of a dedicated DNA binding subdomain and the functional significance of relative subdomain positioning upon substrate binding. These insights provide a framework in which to analyze the native function of pol X. Pol X structure The architecture of the prototypical DNA polymerase domain is likened to a right hand that can grasp DNA (for review see ref. 4), with the subdomains referred to as the fingers, palm, and thumb. The palm subdomain has three carboxylates that bind two catalytically essential metals involved in the nucleotidyl transferase reaction. Crystal structures of DNA polymerases from several polymerase families, excluding pol β, indicate that the palm subdomains are structurally homologous, but the fingers and thumb are not. The thumb and fingers subdomains have primary roles in duplex DNA binding and deoxynucleoside 5′-triphosphate (dNTP) selection, respectively. DNA polymerase β has functionally equivalent subdomains, but the topology of the palm is unique 5. The ASFV pol X shares sequence similarity with the C-terminal region of pol β

The current model for base excision repair (BER) involves two general sub-pathways termed single-nucleotide BER and long patch BER that are distinguished by their repair patch sizes and the enzymes/co-factors involved. Both sub-pathways involve a series of sequential steps from initiation to completion of repair. The BER sub-pathways are designed to sequester the various intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the BER intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In the present study we designed an in vitro assay to explore the question of whether there is a channeling or "hand-off" of the repair intermediates during BER in vitro. The results show that when BER enzymes are pre-bound to the initial single-nucleotide BER intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase β and then to DNA ligase. In the long patch BER subpathway, where the 5'-end of the incised strand is blocked, the intermediate after DNA polymerase β gap filling is not channeled to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.

It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF 2 -substituted nucleotide analogs—one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have “correct” polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF 2 -substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF 2 groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol β) binding pocket by obtaining a 2.5 Å resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate/Mg 2+ interaction over Watson–Crick hydrogen bonding was found and discussed.

An aerobic environment burdens DNA polymerase substrates with oxidized substrates (DNA and nucleotide pools). A major promutagenic lesion resulting from oxidative stress is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG). Guanine oxidation alters the hydrogen bonding properties of the base and glycosidic-preference of the nucleotide. The favored glycosidic syn-conformation exposes the Hoogsteen edge of the base for hydrogen bonding with adenine during DNA synthesis. The cell has recognized the threat of this lesion and has evolved an intricate surveillance system to provide DNA polymerases with unmodified substrates. Failure to do so leads to transversion mutations. Since the mutagenic properties of the base are dictated by the anti-syn-conformation of the nucleotide, the molecular interactions of 8-oxoG in the confines of the DNA polymerase active site are expected to influence its coding potential. Recent structural characterization of DNA polymerases from several families with this lesion in the nascent base pair binding pocket has provided insight to the mutagenic properties of this modified nucleotide. These structures reveal that flexibility around the template-binding pocket can permit 8-oxoG to assume an anti- or syn-conformation and code for cytosine or adenine incorporation, respectively. In contrast, the binding pocket for the incoming nucleotide does not have this flexibility so that 8-oxodGTP insertion opposite cytosine is strongly discouraged.

DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase beta to utilize nucleotides with modified sugars. DNA polymerase beta readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3'-endo sugar pucker facilitates nucleotide insertion, whereas a 2'-endo conformation inhibits insertion.

Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA polymerase (pol) beta, a family X polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol beta insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol beta bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.

The oxidized DNA base 8-oxoguanine (8-oxoG) is implicated in neuronal CAG repeat expansion associated with Huntington disease, yet it is unclear how such a DNA base lesion and its repair might cause the expansion. Here, we discovered size-limited expansion of CAG repeats during repair of 8-oxoG in a wild-type mouse cell extract. This expansion was deficient in extracts from cells lacking pol beta and HMGB1. We demonstrate that expansion is mediated through pol beta multinucleotide gap-filling DNA synthesis during long-patch base excision repair. Unexpectedly, FEN1 promotes expansion by facilitating ligation of hairpins formed by strand slippage. This alternate role of FEN1 and the polymerase beta (pol beta) multinucleotide gap-filling synthesis is the result of uncoupling of the usual coordination between pol beta and FEN1. HMGB1 probably promotes expansion by stimulating APE1 and FEN1 in forming single strand breaks and ligatable nicks, respectively. This is the first report illustrating that disruption of pol beta and FEN1 coordination during long-patch BER results in CAG repeat expansion.

β,γ-Fluoromethylene analogues of nucleotides are generally considered to be useful mimics of the natural substrates for DNA polymerases, but direct structural evidence defining their active site interactions has not been available. In addition, the effect of introducing a new chiral center (the CHF carbon) has been unexplored. We report here structural studies of the diastereomeric β,γ-CHF analogues (R, 3; S, 4) of dGTP interacting with the active site of DNA pol β, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. The conjugation of dGMP 5‘-morpholidate with a tetrabutylammonium salt of (fluoromethylene)bisphosphonic acid (6b, prepared like its difluoro analogue 7b via fluorination of tetraisopropyl methylenebisphosphonate carbanion with Selectfluor) gives a 1:1 mixture of 3 and 4 (by 19F NMR, pH 10). The β,γ-CF2 (2) and β,γ-CH2 (1) dGTP analogues were also synthesized. Crystallization from a solution containing 3 + 4 together with a preformed DNA pol β complex o...

Summary We report the crystallographic structures of DNA polymerase β with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn 2+ . Substituting Mn 2+ for Mg 2+ significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3′ of the primer terminus away from the α-phosphate of the incoming nucleotide, thereby deterring misincorporation.

The mammalian family X DNA polymerases (DNA polymerases beta, lambda, mu, and TdT) contribute to base excision repair and double-strand break repair by virtue of their ability to fill short gaps in DNA. Structural information now exists for all four of these enzymes, making this the first mammalian polymerase family whose structural portrait is complete. Here we consider how distinctive structural features of these enzymes contribute to their biological functions in vivo.

With an increasing number of structural, kinetic, and modeling studies of diverse DNA polymerases in various contexts, a complex dynamical view of how atomic motions might define molecular "gates" or checkpoints that contribute to polymerase specificity and efficiency is emerging. Such atomic-level information can offer insights into rate-limiting conformational and chemical steps to help piece together mechanistic views of polymerases in action. With recent advances, modeling and dynamics simulations, subject to the well-appreciated limitations, can access transition states and transient intermediates along a reaction pathway, both conformational and chemical, and such information can help bridge the gap between experimentally determined equilibrium structures and mechanistic enzymology data. Focusing on DNA polymerase beta (pol beta), we present an emerging view of the geometric, energetic, and dynamic selection criteria governing insertion rate and fidelity mechanisms of DNA polymerases, as gleaned from various computational studies and based on the large body of existing kinetic and structural data. The landscape of nucleotide insertion for pol beta includes conformational changes, prechemistry, and chemistry "avenues", each with a unique deterministic or stochastic pathway that includes checkpoints for selective control of nucleotide insertion efficiency. For both correct and incorrect incoming nucleotides, pol beta's conformational rearrangements before chemistry include a cascade of slow and subtle side chain rearrangements, followed by active site adjustments to overcome higher chemical barriers, which include critical ion-polymerase geometries; this latter notion of a prechemistry avenue fits well with recent structural and NMR data. The chemical step involves an associative mechanism with several possibilities for the initial proton transfer and for the interaction among the active site residues and bridging water molecules. The conformational and chemical events and associated barriers define checkpoints that control enzymatic efficiency and fidelity. Understanding the nature of such active site rearrangements can facilitate interpretation of existing data and stimulate new experiments that aim to probe enzyme features that contribute to fidelity discrimination across various polymerases via such geometric, dynamic, and energetic selection criteria.

Human DNA polymerase beta (pol beta) fills gaps in DNA as part of base excision DNA repair. Due to its small size it is a convenient model enzyme for other DNA polymerases. Its active site contains two Mg(2+) ions, of which one binds an incoming dNTP and one catalyzes its condensation with the DNA primer strand. Simulating such binuclear metalloenzymes accurately but computationally efficiently is a challenging task. Here, we present a magnesium-cationic dummy atom approach that can easily be implemented in molecular mechanical force fields such as the ENZYMIX or the AMBER force fields. All properties investigated here, namely, structure and energetics of both Michaelis complexes and transition state (TS) complexes were represented more accurately using the magnesium-cationic dummy atom model than using the traditional one-atom representation for Mg(2+) ions. The improved agreement between calculated free energies of binding of TS models to different pol beta variants and the experimentally determined activation free energies indicates that this model will be useful in studying mutational effects on catalytic efficiency and fidelity of DNA polymerases. The model should also have broad applicability to the modeling of other magnesium-containing proteins.

Determination of the protonation state of titratable protein residues is of critical importance for the interpretation of active site chemistry, as well as for understanding the role of electrostatic interactions in protein folding and stability. However, protein titration studies are limited by the fact that, at extreme pH values, increasing fractions of unfolded or partially unfolded structures may be present. This problem is particularly acute for lysine residues which have high pK values. In the present study, we point out that the use of the 13C resonance of lysine C-5 as a reporter for titration of the epsilon-amino group is preferable to the use of C-6 due to the 5-fold greater titration shift, so that reasonable results can be obtained using a two parameter fit of data obtained over a more limited pH range. A new synthetic procedure for [5-13C]lysine is described, and the pK value for Lys72 in the lyase domain of DNA polymerase beta has been determined using the [5-13C]lysine-labeled enzyme. The results agree well with recent studies of the Pol lambda lyase domain, demonstrating that the pK value for this residue is not optimized for Schiff base chemistry (Gao et al., Biochemistry 2006, 45, 1785-1794). We also have re-evaluated data for the pK of Lys73 in the TEM-1 beta-lactamase.

DNA polymerase beta plays an essential role in the base excision repair pathway necessary to cleanse the genome of simple base lesions and abasic sites. Abasic sites arise in DNA from spontaneous base loss (depurination) and DNA-damage specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and the damaged base. DNA polymerase beta contributes two enzymatic activities: DNA synthesis and deoxyribose-phosphate removal through nucleotidyl transferase and lyase mechanisms, respectively. The active site for each of these activities resides on a distinct domain of the protein: 31-kDa polymerase domain and amino-terminal 8-kDa lyase domain. The simple organization of each domain and the ability to assay each activity have hastened our understanding of the faithful replication of DNA during repair synthesis and the flux of intermediates through single nucleotide base excision repair and its alternate pathways.

DNA polymerase (pol) β is a small eukaryotic DNA polymerase composed of two domains. Each domain contributes an enzymatic activity (DNA synthesis and deoxyribose phosphate lyase) during the repair of simple base lesions. These domains are termed the polymerase and lyase domains, respectively. Pol β has been an excellent model enzyme for studying the nucleotidyl transferase reaction and substrate discrimination at a molecular level. In this review, recent crystallographic studies of pol β in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA (apurinic sites and 8-oxoguanine) are described. Structures of these catalytic intermediates provide unexpected insights into mechanisms by which DNA polymerases enhance genome stability. These structures also provide an improved framework that permits computational studies to facilitate the interpretation of detailed kinetic analyses of this model enzyme.

DNA polymerase β plays an essential role in the base excision repair pathway necessary to cleanse the genome of simple base lesions and abasic sites. Abasic sites arise in DNA from spontaneous base loss (depurination) and DNA‐damage specific glycosylases that hydrolyze the N‐glycosidic bond between the deoxyribose and the damaged base. DNA polymerase β contributes two enzymatic activities: DNA synthesis and deoxyribose‐phosphate removal through nucleotidyl transferase and lyase mechanisms, respectively. The active site for each of these activities resides on a distinct domain of the protein: 31‐kDa polymerase domain and amino‐terminal 8‐kDa lyase domain. The simple organization of each domain and the ability to assay each activity have hastened our understanding of the faithful replication of DNA during repair synthesis and the flux of intermediates through single nucleotide base excision repair and its alternate pathways.

Molecular dynamics simulations of DNA polymerase (pol) beta complexed with different incorrect incoming nucleotides (G x G, G x T, and T x T template base x incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol beta structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, alpha-helix N of the thumb subdomain believed to be required for pol beta's catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G x C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (P(alpha)-O3') for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., alpha-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol beta-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.

Crystallographic characterization of DNA polymerase beta (pol beta) has suggested that multiple-domain and subdomain motions occur during substrate binding and catalysis. NMR studies of [methyl-(13)C]methionine-labeled pol beta were conducted to characterize the structural and dynamic response to ligand binding. The enzyme contains seven methionine residues, one of which is at the amino terminus and is partially removed by the expression system. Three of the methyl resonances were readily assigned using site-directed mutants. Assignment of the resonances of Met155, Met158, and Met191 was more difficult due to the spatial proximity of these residues, so that assignments were based on NOESY-HSQC data and on the response to paramagnetic Co(2+) addition, as well as shift perturbations observed for the site-directed mutants. The response of the methyl resonances to substrate binding was evaluated by the serial addition of a template oligonucleotide, a downstream 5'-phosphorylated oligonucleotide, and a primer oligonucleotide to create a two-nucleotide-gapped DNA substrate. Addition of the single-stranded template DNA resulted in selective broadening of the methyl resonance of Met18 in the 8 kDa lyase domain, and this resonance then shifted and sharpened upon addition of a 5'-phosphate-terminated downstream complementary oligonucleotide. Conversion of the two-nucleotide-gapped DNA substrate to a single-nucleotide-gapped substrate by incorporation of ddCMP produced a small perturbation of the Met236 resonance, which makes contact with the primer strand in the crystal structure. The addition of a second equivalent of ddCTP to form the pol beta-DNA-ddCTP ternary complex resulted in significant shifts for the resonances corresponding to Met155, Met191, Met236, and Met282. The Met155 methyl resonance is severely broadened, while the Met191 and Met282 resonances exhibit significant but less extreme broadening. Since only Met236 makes contact with the substrate, the effects on Met155, Met236, and Met282 result from indirect conformational and dynamic perturbations. Previous crystallographic characterization of this abortive complex indicated that a polymerase subdomain or segment (alpha-helix N) repositions itself to form one face of the binding pocket for the nascent base pair. Met282 serves as a probe for motion in this segment. Addition of Mg(2+)-dATP to pol beta in the absence of DNA produced qualitatively similar but much smaller effects on Met191 and Met155, but did not strongly perturb Met282, leading to the conclusion that Mg(2+)-dATP alone is insufficient to produce the large conformational changes that are observed in the abortive complex involving the gapped DNA with a blocked primer and ddNTP. Thus, the NMR data indicate that the nucleotide-DNA interaction appears to be essential for conformational activation.

To dissect the effects of the nucleotide-binding and catalytic metal ions on DNA polymerase mechanisms for DNA repair and synthesis, aside from the chemical reaction, we investigate their roles in the conformational transitions between closed and open states and assembly/disassembly of the active site of polymerase beta/DNA complexes before and after the chemical reaction of nucleotide incorporation. Using dynamics simulations, we find that closing before chemical reaction requires both divalent metal ions in the active site while opening after the chemical reaction is triggered by release of the catalytic metal ion. The critical closing is stabilized by the interaction of the incoming nucleotide with conserved catalytic residues (Asp190, Asp192, Asp256) and the two functional magnesium ions; without the catalytic ion, other protein residues (Arg180, Arg183, Gly189) coordinate the incomer's triphosphate group through the nucleotide-binding ion. Because we also note microionic heterogeneity near the active site, Mg(2+) and Na(+) ions can diffuse into the active site relatively rapidly, we suggest that the binding of the catalytic ion itself is not a rate-limiting conformational or overall step. However, geometric adjustments associated with functional ions and proper positioning in the active site, including subtle but systematic motions of protein side chains (e.g., Arg258), define slow or rate-limiting conformational steps that may guide fidelity mechanisms. These sequential rearrangements are likely sensitively affected when an incorrect nucleotide approaches the active site. Our suggestion that subtle and slow adjustments of the nucleotide-binding and catalytic magnesium ions help guide polymerase selection for the correct nucleotide extends descriptions of polymerase pathways and underscores the importance of the delicate conformational events both before and after the chemical reaction to polymerase efficiency and fidelity mechanisms.

To link conformational transitions noted for DNA polymerases with kinetic results describing catalytic efficiency and fidelity, we investigate the role of key DNA polymerase beta residues on subdomain motion through simulations of five single-residue mutants: Arg-283-Ala, Tyr-271-Ala, Asp-276-Val, Arg-258-Lys, and Arg-258-Ala. Since a movement toward a closed state was only observed for R258A, we suggest that Arg(258) is crucial in modulating motion preceding chemistry. Analyses of protein/DNA interactions in the mutant active site indicate distinctive hydrogen bonding and van der Waals patterns arising from compensatory structural adjustments. By comparing closed mutant complexes with the wild-type enzyme, we interpret experimentally derived nucleotide binding affinities in molecular terms: R283A (decreased), Y271A (increased), D276V (increased), and R258A (decreased). Thus, compensatory interactions (e.g., in Y271A with adjacent residues Phe(272), Asn(279), and Arg(283)) increase the overall binding affinity for the incoming nucleotide although direct interactions may decrease. Together with energetic analyses, we predict that R258G might increase the rate of nucleotide insertion and maintain enzyme fidelity as R258A; D276L might increase the nucleotide binding affinity more than D276V; and R283A/K280A might decrease the nucleotide binding affinity and increase misinsertion more than R283A. The combined observations regarding key roles of specific residues (e.g., Arg(258)) and compensatory interactions echo the dual nature of polymerase active site, namely versatility (to accommodate various basepairs) and specificity (for preserving fidelity) and underscore an organized but pliant active site essential to enzyme function.

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/TT/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.

DNA polymerase β (pol β ) plays a crucial role in the base-excision repair (BER) pathway that cleanses the genome of apurinic/apyrimidinic (AP) sites. AP sites arise in DNA from spontaneous base loss (depurination) and DNA damage-specific glycosylases that hydrolyze the N -glycosidic bond between the deoxyribose and damaged base. AP sites represent a potentially dangerous lesion to a cell since they can be mutagenic or cytotoxic. Pol β is composed of two specialized domains that contribute two enzymatic activities, DNA synthesis and lyase, during the repair of AP sites. The amino-terminal domain of pol β possesses the lyase activity necessary to remove the 5′-deoxyribose phosphate intermediate generated during BER. Failure to remove this group may initiate alternate BER pathways. The larger polymerase domain has nucleotidyl transferase activity. This domain has a modular organization with subdomains that bind duplex DNA, catalytic metals, and the correct nucleoside triphosphate in a template-dependent manner. Detailed crystallographic structures of pre-catalytic complexes and mutagenic intermediates of pol β have provided insights into the strategies utilized by polymerases to ensure high-fidelity DNA synthesis.

The large-scale opening motion of mammalian DNA polymerase beta is followed at atomic resolution by dynamic simulations that link crystal "closed" and "open" conformations. The closing/opening conformational change is thought to be key to the ability of polymerases to choose a correct nucleotide (through "induced fit") and hence maintain DNA repair synthesis fidelity. Corroborating available structural and kinetic measurements, our studies bridge static microscopic crystal structures with macroscopic kinetic data by delineating a specific sequence, Phe272 ring flip, large thumb movement, Arg258 rotation with release of catalytic Mg2+, together with estimated time-scales, that suggest the Arg258 rearrangement as a limiting factor of large subdomain motions. If similarly slow in the closing motion, this conformational change might be restricted further when an incorrect nucleotide binds and thus play a role in pol beta's selectivity for the correct nucleotide. These results suggest new lines of experimentation in the study of polymerase mechanisms (e.g. enzyme mutants), which should provide further insights into mechanisms of error discrimination and DNA synthesis fidelity.

Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.

The major product of oxidative base damage is 8-oxo-7,8-dihydro-2′-deoxyguanine (8odG). The coding potential of this lesion is modulated by its glycosidic torsion angle that controls whether its Watson-Crick or Hoogsteen edge is utilized for base pairing. The 2.0 Å structure of DNA polymerase (pol) β bound with 8odGTP opposite template adenine indicates that the modified nucleotide assumes the mutagenic syn-conformation and that the non-mutagenic anti-conformation would be incompatible with efficient DNA synthesis.

DNA polymerase beta, the smallest eukaryotic DNA polymerase, is designed to synthesize DNA in short DNA gaps during DNA repair. It is composed of two specialized domains that contribute essential enzymatic activities to base excision repair (BER). Its amino-terminal domain possesses a lyase activity necessary to remove the 5'-deoxyribose phosphate (dRP) intermediate generated during BER. Removal of the dRP moiety is often the rate-limiting step during BER. Failure to remove this group may initiate alternate BER pathways. The larger polymerase domain has nucleotidyl transferase activity. This domain has a modular organization with sub-domains that bind duplex DNA, catalytic metals, and the correct nucleoside triphosphate in a template-dependent manner. X-ray crystal structures of DNA polymerase beta, with and without bound substrates, has inferred that domain, sub-domain, and substrate conformational changes occur upon ligand binding. Many of these conformational changes are distinct from those observed in structures of other DNA polymerases. This review will examine the structural aspects of DNA polymerase beta that facilitate its role in BER.

The structures of open and closed conformations of DNA polymerase beta (pol beta) suggests that the rate of single-nucleotide deletions during synthesis may be modulated by interactions in the DNA minor groove that align the templating base with the incoming dNTP. To test this hypothesis, we measured the single-base deletion error rates of wild-type pol beta and lysine and alanine mutants of Arg(283), whose side chain interacts with the minor groove edge of the templating nucleotide at the active site. The error rates of both mutant enzymes are increased >100-fold relative to wild-type pol beta. Template engineering experiments performed to distinguish among three possible models for deletion formation suggest that most deletions in repetitive sequences by pol beta initiate by strand slippage. However, pol beta also generates deletions by a different mechanism that is strongly enhanced by the substitutions at Arg(283). Analysis of error specificity suggests that this mechanism involves nucleotide misinsertion followed by primer relocation, creating a misaligned intermediate. The structure of pol beta bound to non-gapped DNA also indicates that the templating nucleotide and its downstream neighbor are out of register in the open conformation and this could facilitate misalignment (dNTP or primer terminus) with the next template base.

The cDNA encoding the human polymerase β from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase β cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase β mRNA in Escherichia coli and all yielded a high level of human polymerase β. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters ( k cat and K m ) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.

Arginine 72 in human immunodeficiency virus type 1 reverse transcriptase (RT), a highly conserved residue among retroviral polymerases and telomerases, forms part of the binding pocket for the nascent base pair. We show here that replacement of Arg72 by alanine strongly alters fidelity in a highly unusual manner. R72A reverse transcriptase is a frameshift and base substitution antimutator polymerase whose increased fidelity results both from increased nucleotide selectivity and from a decreased ability to extend mismatched primer termini. Thus, Arg72-substrate interactions in wild-type human immunodeficiency virus type 1 RT can stabilize incorrect nucleotides allowing misinsertion and promoting extension of mismatched and perhaps misaligned template-primers. In contrast to the higher fidelity at most sites, R72A RT is highly error-prone for misincorporations opposite template T in the sequence context: 5′-CTGG. Surprisingly, this results mostly from a 1200-fold increase in the apparent K m for correct dAMP incorporation. Thus, Arg72 interactions with substrate are critical for the stability of the correct T·dAMP base pair when the 5′-CTGG sequence is present in the binding pocket for the nascent base pair. Collectively, the data show that a mutant polymerase may yield higher than normal average replication fidelity, yet paradoxically place specific sequences at very high risk of mutation.

Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonucleotide was synthesized in situ by DNA polymerases. 5-[N-(2-nitro-5-azidobenzoyl)-trans -3-aminopropenyl-1]deoxyuridine-5'-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer logical ortemplate either by human DNA polymerase alpha primase (polalpha), human DNA polymerase beta (polbeta) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The polymerase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA. The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE. The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA. The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3'-end of the primer, while the p14 subunit locates relatively far from the 3'-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.

Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5' to a DNA abasic site. We have characterized this reaction by steady-state and transient-state kinetics with purified human AP endo, which had been expressed in Escherichia coli. The substrate was a 49-base pair oligonucleotide with an abasic site at position 21. This substrate was generated by treating a 49-mer duplex oligonucleotide with a single G/U located at position 21 with uracil-DNA glycosylase. The enzymatic products of the AP endo nicking reaction were a 20-mer with a hydroxyl group at the 3'-terminus and a 28-mer with a phosphodeoxyribose at the 5'-terminus. To obtain maximal enzymatic activity, it was necessary to stabilize the abasic site during treatment with uracil-DNA glycosylase with a reducing agent. Otherwise, a 20-mer with phosphoribose at the 3'-terminus resulted from beta-elimination. In agreement with others, Km and kcat were 100 nM and 10 s(-1), respectively. Heat treatment of the abasic site-containing 49-mer without enzyme also resulted in conversion to the beta-elimination product. The resultant heat degradation product was an efficient inhibitor of AP endo with a Ki of 30 nM. The enzyme required divalent cation (Mg2+) for activity, but bound substrate DNA in the absence of Mg2+. Electrophoretic mobility shift assays indicated that AP endo bound tightly to DNA containing an abasic site and formed a 1:1 complex at low enzyme concentrations. The association and dissociation rate constants for substrate binding to AP endo were determined by using a challenge assay to follow AP endo-substrate complex formation. Heat degradation product together with heparin served as an effective trap for free enzyme. The results are consistent with a Briggs-Haldane mechanism where k(on) and k(off) are 5 x 10(7) M(-1) s(-1) and 0.04 s(-1), respectively (Kd = 0.8 nM), kcat is 10 s(-1), and product release is very rapid (i.e. k(off,product) >> 10 s(-1)). This scheme is in excellent agreement with the measured steady-state kinetic parameters.

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase beta (beta-pol) were labeled using a photoreactive analog of dCTP, exo-N-[beta-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5'- triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3'-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP cross-linking was increased in the presence of a dideoxyterminated primer-template with RT, but not with beta-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of beta-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase beta are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5'-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (kcat/Km) or fidelity. The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T.dGTP and A.dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase beta and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.

Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the "helix clamp" (Hermann, T., Meier, T., Gotte, M., and Heumann, H. (1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two alpha-helices (alpha H and alpha I) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in alpha H make important interactions with nucleic acid and influence frameshift fidelity. To define the role of alpha I (residues 278-286) during catalytic cycling, we performed systematic site-directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine. Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized. In contrast to alanine mutants of alpha H, alanine substitution in alpha I did not have a significant effect on template.primer (T.P) binding as revealed by a lack of an effect on Km, T.P, Ki for 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, koff, T.P and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of alpha I lowered the apparent activity of every mutant relative to wild-type enzyme. Titration of two mutants exhibiting the lowest activity with T.P (L282A and R284A) demonstrated that these mutant enzymes could bind T.P stoichiometrically and tightly. In contrast, active site concentrations determined from "burst" experiments suggest that the lower activity is due to a smaller populations of enzyme bound productively to T.P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent. In contrast, interactions between several residues of alpha H and the DNA minor groove, 3-5 nucleotides from the 3'-primer terminus, are suggested to be critical for DNA binding and fidelity.

Mild proteolysis of rat DNA polymerase beta (beta-pol) generates an N-terminal 8 kDa domain and a C-terminal 31 kDa domain; the 31 kDa domain is degraded to 6 and 27 kDa fragments by further proteolysis [Kumar, A., Widen, S.G., Williams, K.R., Kedar, P., Karpel, R.L., & Wilson S.H. (1990) J. Biol. Chem. 265, 2124-2131]. In the present study, we found that more vigorous trypsin digestion of the 27 kDa fragment of beta-pol produces 10 and 12 kDa subdomains. Thus, rat beta-pol has four distinct proteolytic fragments of 8, 6, 10, and 12 kDa, extending from the N-terminus to the C-terminus, respectively. To map the location of the dNTP binding site(s), intact beta-pol was photoaffinity labeled with 8-azido-ATP or 5-azido-dUTP in presence or absence of competitor dNTP (dATP). The labeled enzyme was subjected to controlled proteolysis, and the resulting labeled peptides were separated and sequenced. Competition with dATP showed that three regions of beta-pol in solution combine to form the dNTP binding pocket as follows: residues 4-40 of the 8 kDa domain; residues 142-206 of the 10 kDa subdomain; and residues 263-280 of the 12 kDa subdomain (alpha-helices M and N). These results are discussed in light of the recent crystal structure of dATP bound to rat beta-pol.