In vitro ligation of oligodeoxynucleotides containing C8-oxidized purine lesions using bacteriophage T4 DNA ligase

Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3' end of the nick compared to the 5' end and to lesions located in the intact template strand. In general, substrates containing the OG.A mismatch were more readily ligated than those with the OG.C mismatch. Ligations of duplexes containing the OA.T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI.A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG.A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG.C-, OI.C-, OI.A-, and OA.T-containing duplexes compared to that of a G.C-containing duplex. Substrates containing Gh.A, Gh.G, Sp.A, and Sp.G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G.C base pair, while the Km value for OG.A was 5 times lower than that for G.C.     

Removal of hydantoin products of 8-oxoguanine oxidation by the Escherichia coli DNA repair enzyme, FPG

An intriguing feature of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is that it is highly reactive toward further oxidation. Indeed, OG has been shown to be a "hot spot" for oxidative damage and susceptible to oxidation by a variety of cellular oxidants. Recent work has identified two new DNA lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), resulting from one-electron oxidation of OG. The presence of Gh and Sp lesions in DNA templates has been shown to result in misinsertion of G and A by DNA polymerases, and therefore, both are potentially mutagenic DNA lesions. The base excision repair (BER) glycosylases Fpg and MutY serve to prevent mutations associated with OG in Escherichia coli, and therefore, we have investigated the ability of these two enzymes to process DNA duplex substrates containing the further oxidized OG lesions, Gh and Sp. The Fpg protein, which removes OG and a variety of other oxidized purine base lesions, was found to remove Gh and Sp efficiently opposite all four of the natural DNA bases. The intrinsic rate of damaged base excision by Fpg was measured under single-turnover conditions and was found to be highly dependent upon the identity of the base opposite the OG, Gh, or Sp lesion; as expected, OG is removed more readily from an OG:C- than an OG:A-containing substrate. However, when adenine is paired with Gh or Sp, the rate of removal of these damaged lesions by Fpg was significantly increased relative to the rate of removal of OG from an OG:A mismatch. The adenine glycosylase MutY, which removes misincorporated A residues from OG:A mismatches, is unable to remove A paired with Gh or Sp. Thus, the activity of Fpg on Gh and Sp lesions may dramatically influence their mutagenic potential. This work suggests that, in addition to OG, oxidative products resulting from further oxidation of OG should be considered when evaluating oxidative DNA damage and its associated effects on DNA mutagenesis.     

Recognition and removal of oxidized guanines in duplex DNA by the base excision repair enzymes hOGG1, yOGG1, and yOGG2

8-Oxo-7,8-dihydroguanine (OG) is susceptible to further oxidation in vitro to form two secondary oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). Previous work from this laboratory has shown that OG, Gh, and Sp are recognized and excised from duplex DNA substrates by the Escherichia coli DNA repair enzyme Fpg. In this report, we extend these studies to the functionally related eukaryotic OG glycosylases (OGG) from yeast and humans: yOGG1, yOGG2, and hOGG1. The hOGG1 enzyme was active only toward the removal of 8-oxoguanine, exhibiting a 1000-fold faster rate of removal of 8-oxoguanine from OG.C-containing duplexes relative to their OG.A counterparts. Duplexes containing Gh or Sp opposite any of the four natural bases were not substrates for the hOGG1 enzyme. In contrast, both yOGG1 and yOGG2 enzymes removed Gh and Sp in a relatively efficient manner from an 18 bp duplex. No significant difference was observed in the rate of reaction of Gh- and Sp-containing duplexes with yOGG1. However, yOGG2 removed Sp at a faster rate than Gh. Both yOGG enzymes exhibit a negligible dependence on the base opposite the lesion, suggesting that the activity of these enzymes may be promutagenic. Surprisingly, in the 18 bp sequence context, both yOGG enzymes did not exhibit OG removal activity. However, both removed OG in a 30 bp duplex with a different sequence surrounding the OG. The wide range of repair efficiencies observed by these enzymes with different substrates in vitro suggests that this could greatly affect the mutagenicity of these lesions in vivo. Indeed, the greater efficiency of the yOGG proteins for removal of the further oxidized products, Gh and Sp, over their 8-oxoguanine parent, suggests that these lesions may be the preferred substrates in vivo.     

Effect of the oxidized guanosine lesions spiroiminodihydantoin and guanidinohydantoin on proofreading by Escherichia coli DNA polymerase I (Klenow fragment) in different sequence contexts

Oxidative damage to DNA by endogenous and exogenous reactive oxygen species has been directly linked to cancer, aging, and a variety of neurological disorders. The potential mutagenicity of the primary guanine oxidation product 8-oxo-7,8-dihydroguanine (Og) has been studied intensively, and much information is available about its miscoding potential in vitro and in vivo. Recently, a variety of DNA lesions have been identified as oxidation products of both guanine and 8-oxoguanine, among them spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). To address questions concerning the mutagenic potential of these secondary products of guanine oxidation, the effect of the lesions on proofreading by DNA polymerase was studied in vitro using the Klenow fragment of Escherichia coli polymerase I (Kf exo+). For the first time, k(cat)/K(m) values were obtained for proofreading of the X:N mismatches (X = Og, Gh, or Sp; N = A, G, or C). Proofreading studies of the terminal mismatches demonstrated the significance of the sequence context flanking the lesion on the 3' side. In addition, a sequence dependence was observed for Gh based on the identity of the base on the 5' side of the lesion providing evidence for a primer slippage mode if N was complementary to the 5' base. Internal mismatches were recognized by Kf exo+ resulting in the excision of the correct base pairs flanking mismatches from the 5' side. The absence of a sequence effect for the Gh- and Sp-containing duplexes can be attributed to the severe destabilization of the lesion-containing duplexes that promotes interaction with the exonuclease domain of the Klenow fragment.     

Unusual structural features of hydantoin lesions translate into efficient recognition by Escherichia coli Fpg

Oxidation of guanine (G) and 8-oxoguanine (OG) with a wide variety of oxidants yields the hydantoin lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). These two lesions have garnered much recent attention due to their unusual structures and high mutagenic potential. We have previously shown that duplexes containing Gh and Sp are substrates for the base excision repair glycosylase Escherichia coli Fpg (EcFpg). To evaluate the recognition features of these unusual lesions, binding and footprinting experiments were performed using a glycosylase inactive variant, E3Q EcFpg, and 30 bp duplexes containing the embedded lesions. Surprisingly, E3Q EcFpg was found to bind significantly more tightly ( approximately 1000-fold) to duplexes containing Gh or Sp over the corresponding duplexes containing OG. This may be a consequence of the helix-destabilizing nature of the hydantoin lesions that facilitates their recognition within duplex DNA. Though DNA binding affinities of E3Q EcFpg with Gh- and Sp-containing duplexes were found to be similar to each other, hydroxyl radical footprinting using methidium-propyl-EDTA (MPE)-Fe(II) revealed subtle differences between binding of E3Q EcFpg to the two lesions. Most notably, in the presence of E3Q EcFpg, the Sp nucleotide (nt) is hyperreactive toward cleavage by MPE-Fe(II)-generated hydroxyl radicals, suggestive of the formation of an intercalation site for the MPE-Fe(II) reagent at the Sp nt. Interestingly, increasing the duplex length from 18 to 30 bp enhanced the excision efficiency of Gh and Sp paired with C, G, or T by EcFpg such that these substrates are processed as efficiently as the signature substrate lesion, OG. Moreover, the base removal activity with these two lesions was more efficient than removal of OG when in a base pairing context opposite A. The high affinity and efficient activity of EcFpg toward the hydantoin lesions suggest that EcFpg mediates repair of the lesions in vivo. Notably, the facile activity of EcFpg toward Gh and Sp in base pairing contexts with G and A, which are likely to be present after DNA replication, would be detrimental and enhance mutagenesis.     

Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2

8-Oxoguanine (8-oxoG) is an unstable mutagenic DNA lesion that is prone to further oxidation. High valent metals such as Cr(V) and Ir(IV) readily oxidize 8-oxoG to form guanidinohydantoin (Gh), its isomer iminoallantoin (Ia), and spiroiminodihydantoin (Sp). When present in DNA, these lesions show enhanced base misincorporation over the parent 8-oxoG lesion leading to G --> T and G --> C transversion mutations and polymerase arrest. These findings suggested that further oxidized lesions of 8-oxoG are more mutagenic and toxic than 8-oxoG itself. Repair of oxidatively damaged bases, including Sp and Gh/Ia, are initiated by the base excision repair (BER) system that involves the DNA glycosylases Fpg, Nei, and Nth in E. coli. Mammalian homologs of two of these BER enzymes, OGG1 and NTH1, have little or no affinity for Gh/Ia and Sp. Herein we report that two recently identified mammalian glycosylases, NEIL1 and NEIL2, showed a high affinity for recognition and cleavage of DNA containing Gh/Ia and Sp lesions. NEIL1 and NEIL2 recognized both of these lesions in single-stranded DNA and catalyzed the removal of the lesions through a beta- and delta-elimination mechanism. NEIL1 and NEIL2 also recognized and excised the Gh/Ia lesion opposite all four natural bases in double-stranded DNA. NEIL1 was able to excise the Sp lesion opposite the four natural bases in double-stranded DNA, however, NEIL2 showed little cleavage activity against the Sp lesion in duplex DNA although DNA trapping studies show recognition and binding of NEIL2 to this lesion. This work suggests that NEIL1 and NEIL2 are essential in the recognition of further oxidized lesions arising from 8-oxoG and implies that these BER glycosylases may play an important role in the repair of DNA damage induced by carcinogenic metals.     

Human DNA polymerase iota promiscuous mismatch extension

Human DNA polymerase iota is a low-fidelity template copier that preferentially catalyzes the incorporation of the wobble base G, rather than the Watson-Crick base A, opposite template T (Tissier, A., McDonald, J. P., Frank, E. G., and Woodgate, R. (2000) Genes Dev. 14, 1642-1650; Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S., and Prakash, L. (2000) Nature 406, 1015-1019; Zhang, Y., Yuan, F., Wu, X., and Wang, Z. (2000) Mol. Cell. Biol. 20, 7099-7108). Here, we report on its ability to extend all 12 possible mispairs and 4 correct pairs in different sequence contexts. Extension from both matched and mismatched primer termini is generally most efficient and accurate when A is the next template base. In contrast, extension occurs less efficiently and accurately when T is the target template base. A striking exception occurs during extension of a G:T mispair, where the enzyme switches specificity, "preferring" to make a correct A:T base pair immediately downstream from an originally favored G:T mispair. Polymerase iota generates a variety of single and tandem mispairs with high frequency, implying that it may act as a strong mutator when copying undamaged DNA templates in vivo. Even so, its limited ability to catalyze extension from a relatively stable primer/template containing a "buried" mismatch suggests that polymerase iota-catalyzed errors are confined to short template regions.     

Conformations of DNA Duplexes Containing 8-Oxoguanine

Abstract

As a step towards elucidating the mechanisms of mutagenesis induced by irradiation and oxidation, we study the incorporation of 8-oxoguanine (OG) into duplex DNA. Molecular modelling is used to reveal changes in DNA conformational parameters due to mispairs within the sequences d(A₅XA₅) · d(T₅YT₅) and d(G₅XG₅) · d(C₅YC₅) where one of the bases of the bases of the central X:Y pair is OG and the other A T, G or C. The G:C to OG:C replacements in DNA duplexes produce only minor conformational changes, similar to normal base sequence effects. The calculations suggest that both OG(syn): G and OG(syn): A mispairs can also be introduced without drastic distortion of sugar-phosphate backbone. The distortions produced by OG-containing mispairs are also found to be sequence dependent Overall these calculations suggest that the G→OG conversion could be an important factor in the irradiative or oxidative damage of DNA.     

Loss of DNA minor groove interactions by exonuclease-deficient Klenow polymerase inhibits O6-methylguanine and abasic site translesion synthesis

The importance of DNA polymerase-DNA minor groove interactions on translesion synthesis (TLS) was examined in vitro using variants of exonuclease-deficient Klenow polymerase and site-specifically modified DNA oligonucleotides. Polymerase variant R668A lacks primer strand interactions, while variant Q849A lacks template strand interactions. O(6)-Methylguanine (m6G) and abasic site TLS was examined in three stages: dNTP insertion opposite the lesion, extension from a terminal lesion-containing base pair, and the dissociation equilibrium of the polymerase from the lesion-containing template. Less than 5% TLS was observed at the insertion step for either variant on the lesion-containing templates. While extensive TLS was observed for WT polymerase on the m6G template, only incorporation opposite the lesion was observed for the R668A variant. Loss of the template strand interaction, Q849A, resulted in the inability to insert dNTPs opposite either the m6G or abasic lesion. For both variants, extension of purine-containing m6G primer-templates was increased relative to WT polymerase. We observed similar extension efficiencies for all variants, relative to WT, using abasic template-primers. Polymerase dissociation/reassociation was studied through the use of a competitor primer/template complex. Dissociation for WT polymerase increased 2-fold and 3-fold, respectively, for m6G and abasic lesion-containing templates, relative to the natural template. Variants lacking DNA minor groove interactions displayed increased dissociation from DNA templates, relative to WT polymerase, but do not display an increased level of lesion-induced polymerase dissociation. Our results indicate that the primer and template strand interactions of the Klenow polymerase with the DNA minor groove are critical for maintaining the DNA-polymerase complex during translesion synthesis.     

Dimerization of the Klenow fragment of Escherichia coli DNA polymerase I is linked to its mode of DNA binding

Upon associating with a proofreading polymerase, the nascent 3' end of a DNA primer/template has two possible fates. Depending upon its suitability as a substrate for template-directed extension or postsynthetic repair, it will bind either to the 5'-3' polymerase active site, yielding a polymerizing complex, or to the 3'-5' exonuclease site, yielding an editing complex. In this investigation, we use a combination of biochemical and biophysical techniques to probe the stoichiometry, thermodynamic, and kinetic stability of the polymerizing and editing complexes. We use the Klenow fragment of Escherichia coli DNA polymerase I (KF) as a model proofreading polymerase and oligodeoxyribonucleotide primer/templates as model DNA substrates. Polymerizing complexes are produced by mixing KF with correctly base paired (matched) primer/templates, whereas editing complexes are produced by mixing KF with multiply mismatched primer/templates. Electrophoretic mobility shift titrations carried out with matched and multiply mismatched primer/templates give rise to markedly different electrophoretic patterns. In the case of the matched primer/template, the KF.DNA complex is represented by a slow moving band. However, in the case of the multiply mismatched primer/template, the complex is predominantly represented by a fast moving band. Analytical ultracentrifugation measurements indicate that the fast and slow moving bands correspond to 1:1 and 2:1 KF.DNA complexes, respectively. Fluorescence anisotropy titrations reveal that KF binds with a higher degree of cooperativity to the matched primer/template. Taken together, these results indicate that KF is able to dimerize on a DNA primer/template and that dimerization is favored when the first molecule is bound in the polymerizing mode, but disfavored when it is bound in the editing mode. We suggest that self-association of the polymerase may play an important and as yet unexplored role in coordinating high-fidelity DNA replication.     

Positive correlation between DNA polymerase alpha-primase pausing and mutagenesis within polypyrimidine/polypurine microsatellite sequences

Microsatellite DNA sequences are ubiquitous in the human genome, and mutation rates of these repetitive sequences vary with respect to DNA sequence as well as length. We have analyzed polymerase-DNA interactions as a function of microsatellite sequence, using polypyrimidine/polypurine di- and tetranucleotide alleles representative of those found in the human genome. Using an in vitro primer extension assay and the mammalian DNA polymerase alpha-primase complex, we have observed a polymerase termination profile for each microsatellite that is unique to that allele. Interestingly, a periodic termination profile with an interval size (9-11 nucleotides) unrelated to microsatellite unit length was observed for the [TC](20) and [TTCC](9) templates. In contrast, a unit-punctuated polymerase termination profile was found for the longer polypurine templates. We detected strong polymerase pauses within the [TC](20) allele at low reaction pH which were eliminated by the addition of deaza-dGTP, consistent with these specific pauses being a consequence of triplex DNA formation during DNA synthesis. Quantitatively, a strand bias was observed in the primer extension assay, in that polymerase synthesis termination is more intense when the polypurine sequence serves as the template, relative to its complementary polypyrimidine sequence. The HSV-tk forward mutation assay was utilized to determine the corresponding polymerase alpha-primase error frequencies and specificities at the microsatellite alleles. A higher microsatellite polymerase error frequency (50x10(-4) to 60x10(-4)) was measured when polypurine sequences serve as templates for DNA synthesis, relative to the polypyrimidine template (18x10(-4)). Thus, a positive correlation exists between polymerase alpha-primase pausing and mutagenesis within microsatellite DNA alleles.     

Repair of hydantoins, one electron oxidation product of 8-oxoguanine, by DNA glycosylases of Escherichia coli

8-Oxoguanine (8-oxoG), induced by reactive oxygen species and arguably one of the most important mutagenic DNA lesions, is prone to further oxidation. Its one-electron oxidation products include potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) because of their mispairing with A or G. All three oxidized base-specific DNA glycosylases of Escherichia coli, namely endonuclease III (Nth), 8-oxoG-DNA glycosylase (MutM) and endonuclease VIII (Nei), excise Gh and Sp, when paired with C or G in DNA, although Nth is less active than the other two. MutM prefers Sp and Gh paired with C (k_cat/K_m of 0.24–0.26 min^–1 nM^–1), while Nei prefers G over C as the complementary base (k_cat/K_m – 0.15–0.17 min^–1 nM^–1). However, only Nei efficiently excises these paired with A. MutY, a 8-oxoG·A(G)-specific A(G)-DNA glycosylase, is inactive with Gh(Sp)·A/G-containing duplex oligonucleotide, in spite of specific affinity. It inhibits excision of lesions by MutM from the Gh·G or Sp·G pair, but not from Gh·C and Sp·C pairs. In contrast, MutY does not significantly inhibit Nei for any Gh(Sp) base pair. These results suggest a protective function for MutY in preventing mutation as a result of A (G) incorporation opposite Gh(Sp) during DNA replication.     

Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from γ-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k_obs of over 2500 min^?1.     

trans-Lesion synthesis past bulky benzo[a]pyrene diol epoxide N2-dG and N6-dA lesions catalyzed by DNA bypass polymerases

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed. Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.     

Analysis of non-template-directed nucleotide addition and template switching by DNA polymerase

DNA polymerases use an uninterrupted template strand to direct synthesis of DNA. However, some DNA polymerases can synthesize DNA across two discontinuous templates by binding and juxtaposing them, resulting in synthesis across the junction. Primer/template duplexes with 3' overhangs are especially efficient substrates, suggesting that DNA polymerases use the overhangs as regions of microhomology for template synapsis. The formation of these overhangs may be the result of non-template-directed nucleotide addition by DNA polymerases. To examine the relative magnitude and mechanism of template switching, we studied the in vitro enzyme kinetics of template switching and non-template-directed nucleotide addition by the 3'-5' exonuclease-deficient large fragment of Escherichia coli DNA polymerase I. Non-template-directed nucleotide addition and template switching were compared to that of standard primer extension. We found that non-template-directed nucleotide addition and template switching showed similar rates and were approximately 100-fold slower than normal template-directed DNA synthesis. Furthermore, non-template-directed nucleotide addition showed a 10-fold preference for adding dAMP to the ends of DNA over that of the other three nucleotides. For template switching, kinetic analysis revealed that the two template substrates acted as a random bireactant system with mixed-type inhibition of substrate binding by one substrate over the other. These data are the first to establish the binding kinetics of two discontinuous DNA substrates to a single DNA polymerase. Our results suggest that although the activities are relatively weak, non-template-directed nucleotide addition and template switching allow DNA polymerases to overcome breaks in the template strand in an error-prone manner.     

Influence of DNA structure on DNA polymerase beta active site function: extension of mutagenic DNA intermediates

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/T > T/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.     

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)_n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.