Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex

Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a DNA oligomer duplex. NMR and energy minimization studies were performed on the 1,N2-propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (C1-A2-T3-G4-X5-G6-T7-A8-C9).(G10-T11-A12-C-13-F14-C15 -A16-T17-G-18) X.F 9-mer duplex. Two-dimensional NMR experiments establish that the X.F 9-mer helix is right-handed with Watson-Crick A.T and G.C base pairing on either side of the lesion site. NOEs are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G4.C15 and G6.C13 which flank the lesion site, as well as to the H1' and H1" protons of the cross strand F14 tetrahydrofuran moiety. These NMR results establish that the exocyclic adduct X5 is positioned between flanking G4.C15 and G6.C13 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,N2-propanodeoxyguanosine adduct fits into the cavity generated by the abasic site.     

Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4'-oxidized abasic sites

A 4'-oxidized abasic site (X) has been synthesized in a defined duplex DNA sequence, 5'-d(CCAAAGXACCGGG)-3'/3'-d(GGTTTCATGGCCC)-5' (1). Its structure has been determined by two-dimensional NMR methods, molecular modeling, and molecular dynamics simulations. 1 is globally B-form with the base (A) opposite X intrahelical and well-stacked. Only the alpha anomer of X is observed, and the abasic site deoxyribose is largely intrahelical. These results are compared with a normal abasic site (Y) in the same sequence context (2). Y is composed of a 60:40 mixture of alpha and beta anomers (2alpha and 2beta). In both 2alpha and 2beta, the base (A) opposite Y is intrahelical and well-stacked and the abasic site deoxyribose is predominantly extrahelical, consistent with the reported structures of the normal abasic site in a similar sequence context [Hoehn, S. T., Turner, C. J., and Stubbe, J. (2001) Nucleic Acids Res. 29, 3413-3423]. Molecular dynamics simulations reveal that the normal abasic site appears to be conformationally more flexible than the 4'-oxidized abasic site. The importance of the structure and flexibility of the abasic site in the recognition by the DNA repair enzyme Ape1 is discussed.     

Solution structure of duplex DNA containing an extrahelical abasic site analog determined by NMR spectroscopy and molecular dynamics.

Translesional DNA synthesis past abasic sites proceeds with the preferential incorporation of dAMP opposite the lesion and, depending on the sequence context, one or two base deletions. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of a DNA heteroduplex containing a synthetic abasic site (tetrahydrofuran) residue positioned in a sequence that promotes one base deletions. Analysis of NMR spectra indicates that the stem region of the duplex adopts a right-handed helical structure and the glycosidic torsion angle is in anti orientation for all residues. NOE interactions establish Watson-Crick alignments for all canonical base pairs of the duplex. Measurement of distance interactions at the lesion site shows the abasic residue excluded from the helix. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. These structures show a regular duplex region and a slight bend at the lesion site. The tetrahydrofuran residue extrudes from the helix and is highly flexible. The model reported here, in conjunction with a previous study performed on abasic sites, explains the structural bias of one-base deletion mutations.     

Effects of base damages on DNA replication--mechanism of preferential purine nucleotide insertion opposite abasic site in template DNA.

DNA polymerase preferentially inserts purine nucleotides opposite non-instructive lesions such as abasic sites during DNA replication. In order to elucidate the mechanism of the preferential insertion, a DNA template containing a model abasic site and primers containing 4 different nucleotides (A,G,C,T) at primer terminus were synthesized. The stability of the primer terminus nucleotide placed opposite the abasic site was evaluated on the basis of its sensitivity to 3'-5' exonuclease associated with DNA polymerase.     

Amino acid templating mechanisms in selection of nucleotides opposite abasic sites by a family a DNA polymerase

Cleavage of the N-glycosidic bond that connects the nucleobase to the backbone in DNA leads to abasic sites, the most frequent lesion under physiological conditions. Several DNA polymerases preferentially incorporate an A opposite this lesion, a phenomenon termed "A-rule." Accordingly, KlenTaq, the large fragment of Thermus aquaticus DNA polymerase I, incorporates a nucleotide opposite an abasic site with efficiencies of A > G > T > C. Here we provide structural insights into constraints of the active site during nucleotide selection opposite an abasic site. It appears that these confines govern the nucleotide selection mainly by interaction of the incoming nucleotide with Tyr-671. Depending on the nucleobase, the nucleotides are differently positioned opposite Tyr-671 resulting in different alignments of the functional groups that are required for bond formation. The distances between the α-phosphate and the 3'-primer terminus increases in the order A < G < T, which follows the order of incorporation efficiency. Additionally, a binary KlenTaq structure bound to DNA containing an abasic site indicates that binding of the nucleotide triggers a remarkable rearrangement of enzyme and DNA template. The ability to resolve the stacking arrangement might be dependent on the intrinsic properties of the respective nucleotide contributing to nucleotide selection. Furthermore, we studied the incorporation of a non-natural nucleotide opposite an abasic site. The nucleotide was often used in studying stacking effects in DNA polymerization. Here, no interaction with Tyr-761 as found for the natural nucleotides is observed, indicating a different reaction path for this non-natural nucleotide.     

NMR characterization of clustered bistrand abasic site lesions: effect of orientation on their solution structure

A unique characteristic of ionizing radiation and radiomimetic anticancer drugs is the induction of clustered damage: two or more DNA lesions (oxidized bases, abasic sites, or strand breaks) occurring in the same or different strands of the DNA molecule within a single turn of the helix. In spite of arising at a lower frequency than single lesions, clustered DNA damage represents an exotic challenge to the repair systems present in the cells and, in some cases, these lesions may escape detection and/or processing. To understand the structural properties of clustered DNA lesions we have prepared two oligodeoxynucleotide duplexes containing adjacent tetrahydrofuran residues (abasic site analogues), positioned one in each strand of the duplex in a 5' or 3' orientation, and determined their solution structure by NMR spectroscopy and molecular dynamics simulations. The NMR data indicate that both duplex structures are right-handed helices of high similarity outside the clustered damage site. The thermal stability of the duplexes is severely reduced by the presence of the abasic residues, especially in a 5' orientation where the melting temperature is 5 degrees C lower. The structures show remarkable differences at the lesion site where the extrahelical location of the tetrahydrofuran residues in the (AP)(2)-5'-staggered duplex contrasts with their smooth alignment along the sugar-phosphate backbone in the (AP)(2)-3'-staggered duplex.     

NMR solution structures of bistranded abasic site lesions in DNA

Ionizing radiation produces clustered lesions in DNA. Since the orientation of bistranded lesions affects their recognition by DNA repair enzymes, clustered damages are more difficult to process and thus more toxic than single oxidative lesions. In order to understand the structural determinants that lead to differential recognition, we used NMR spectroscopy and restrained molecular dynamics to solve the structure of two DNA duplexes, each containing two stable abasic site analogues positioned on opposite strands of the duplex and staggered in the 3' (-1 duplex, (AP) 2-1 duplex) or 5' (+1 duplex, (AP) 2+1 duplex) direction. Cross-peak connectivities observed in the nonexchangeable NOESY spectra indicate compression of the helix at the lesion site of the duplexes, resulting in the formation of two abasic bulges. The exchangeable proton spectra show the AP site partner nucleotides forming interstrand hydrogen bonds that are characteristic of a Watson-Crick G.C base pairs, confirming the extra helical nature of the AP residues. Restrained molecular dynamics simulations generate a set of converging structures in full agreement with the spectroscopic data. In the (AP) 2-1 duplex, the extra helical abasic site residues reside in the minor groove of the helix, while they appear in the major groove in the (AP) 2+1 duplex. These structural differences are consistent with the differential recognition of bistranded abasic site lesions by human AP endonuclease.     

Novel PCR-mediated mutagenesis employing DNA containing a natural abasic site as a template and translesional Taq DNA polymerase

We describe a novel method of PCR-mediated mutagenesis employing DNA containing a natural abasic site and translesional Taq DNA polymerase. This method incorporated an adenine (80.8%) or guanine (7.7%) residue or led to a base deletion mutation (11.2%) opposite the abasic site. We conclude that the combination of DNA containing an abasic site and translesional Taq DNA polymerase is an easy and useful technique for PCR-mediated mutagenesis, having advantages different from those of conventional error-prone PCR.     

DNA synthesis across an abasic lesion by yeast REV1 DNA polymerase

Abasic (apurinic/apyrimidinic) sites are among the most abundant DNA lesions in humans, and they present a strong block to replication. They are also highly mutagenic because when replicative DNA polymerases manage to insert a nucleotide opposite the lesion, they prefer to insert an A. Rev1, a member of Y-family DNA polymerases, does not obey the A-rule. This enzyme inserts a C opposite an abasic lesion with much greater catalytic efficiency than an A, G, or T. We present here the structure of yeast Rev1 in ternary complex with DNA containing an abasic lesion and with dCTP as the incoming nucleotide. The structure reveals a mechanism of synthesis across an abasic lesion that differs from that in other polymerases. The lesion is driven to an extrahelical position, and the incorporation of a C is mediated by an arginine (Arg324) that is conserved in all known orthologs of Rev1, including humans. The hydrophobic cavity that normally accommodates the unmodified G is instead filled with water molecules. Since Gs are especially prone to depurination through a spontaneous hydrolysis of the glycosidic bond, the ability of Rev1 to stabilize an abasic lesion in its active site and employ a surrogate arginine to incorporate a C provides a unique means for the “error-free” bypass of this noninstructional lesion.     

DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5′-CCA AAG₆ XA₈C CGG G-3′, where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4′-oxidized abasic sites. Biochemistry, 46, 3096–3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3′ to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.     

Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNA

The formamidopyrimidine-DNA glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60 degrees bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8-oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.     

Effects of the presence of an aldehydic abasic site on the thermal stability and rates of helix opening and closing of duplex DNA.

The presence of an abasic site in duplex DNA lowers the thermodynamic stability, as monitored by the optical melting temperature, and decreases the rate of imino proton exchange with water, by about an order of magnitude, as monitored by direct measurement of both the exchange lifetimes and the imino proton T1S. The exchange lifetimes of the imino protons with water as a function of base catalyst concentration were analyzed to determine the origin of the effect of the abasic site on imino exchange lifetimes. Analysis of the results showed that the helix opening rate is not significantly changed by the presence of an abasic site. The differences in exchange lifetimes are attributed to a faster helix closing rate in the presence of an abasic site. The faster rate of helix closing may be an important contribution to the stability of abasic sites in duplex DNA to base-catalyzed elimination reaction. It is noted that duplex DNAs containing analogues of the aldehydic abasic site apparently do not exhibit these exchange lifetime effects.     

Yeast Rev1 protein is a G template-specific DNA polymerase

Rev1 protein of Saccharomyces cerevisiae functions with DNA polymerase zeta in mutagenic trans-lesion synthesis. Because of the reported preferential incorporation of a C residue opposite an abasic site, Rev1 has been referred to as a deoxycytidyltransferase. Here, we use steady-state kinetics to examine nucleotide incorporation by Rev1 opposite undamaged and damaged template residues. We show that Rev1 specifically inserts a C residue opposite template G, and it is approximately 25-, 40-, and 400-fold less efficient at inserting a C residue opposite an abasic site, an O(6)-methylguanine, and an 8-oxoguanine lesion, respectively. Rev1 misincorporates G, A, and T residues opposite template G with a frequency of approximately 10(-3) to 10(-4). Consistent with this finding, Rev1 replicates DNA containing a string of Gs in a template-specific manner, but it has a low processivity incorporating 1.6 nucleotides per DNA binding event on the average. From these observations, we infer that Rev1 is a G template-specific DNA polymerase.     

Human DNA polymerase beta mutations allowing efficient abasic site bypass

The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.     

In vitro effects of a C4'-oxidized abasic site on DNA polymerases

Oxidative damage to DNA produces abasic sites resulting from the formal hydrolysis of the nucleotides' glycosidic bonds, along with a variety of oxidized abasic sites. The C4'-oxidized abasic site (C4-AP) is produced by several DNA-damaging agents. This lesion accounts for approximately 40% of the DNA damage produced by bleomycin. The effect of a C4'-oxidized abasic site incorporated at a defined site in a template was examined on Klenow fragments with and without 3' --> 5' exonuclease activity. Both enzymes preferentially incorporated dA > dG > dC, T opposite C4-AP. Neither enzyme is able to extend the primer past the lesion. Experiments with regular AP sites in an otherwise identical template indicate that Klenow does not differentiate between these two disparate abasic sites. Extension of the primer by alternative polymerases pol II, pol II exo(-), pol IV, and pol V was examined. Pol II exo(-) was most efficient. Qualitative translesion synthesis experiments showed that pol II exo(-) preferentially incorporates T opposite C4-AP, followed in order by dG, dA, and dC. Thymidine incorporation opposite C4'-AP is distinct from the pol II exonuclease interaction with a regular AP site in an otherwise identical template. These in vitro experiments suggest that bypass polymerases may play a crucial role in survival of cells in which C4-AP is produced, and unlike a typical AP site, the C4-AP lesion may not follow the "A-rule". The interaction between bypass polymerases and a C4-AP lesion could explain the high levels of G:C --> T:A transversions in cells treated with bleomycin.     

Pre-steady state kinetic studies show that an abasic site is a cognate lesion for the yeast Rev1 protein

Rev1 is a eukaryotic DNA polymerase that rescues replication forks stalled at sites of DNA damage by inserting nucleotides opposite the damaged template bases. Yeast genetic studies suggest that Rev1 plays an important role in rescuing replication forks stalled at one of the most common forms of DNA damage, an abasic site; however, steady state kinetic studies suggest that an abasic site acts as a significant block to nucleotide incorporation by Rev1. Here we examined the pre-steady state kinetics of nucleotide incorporation by yeast Rev1 with damaged and non-damaged DNA substrates. We found that yeast Rev1 is capable of rapid nucleotide incorporation, but only a small fraction of the protein molecules possessed this robust activity. We characterized the nucleotide incorporation by the catalytically robust fraction of yeast Rev1 and found that it efficiently incorporated dCTP opposite a template abasic site under pre-steady state conditions. We conclude from these studies that the abasic site is a cognate lesion for Rev1.     

Mechanism of mutation on DNA templates containing synthetic abasic sites: study with a double strand vector.

Mutagenesis at abasic sites was investigated in E.coli and simian kidney (COS) cells using a duplex shuttle vector containing synthetic analogs of deoxyribose on the phosphodiester backbone. Lesions were positioned on opposite strands of the vector. When the tetrahydrofuranyl analog was used as the abasic site, AT or TA pairs (65-80%) were introduced at the site of the bistrand lesion. Mutagenesis occurred in the absence of SOS induction. Single base deletions (> 80%) dominated the mutational spectra for propanyl and ethanyl analogs of abasic sites lacking a ring structure. For all abasic site analogs, a small proportion of G/C and C/G pairs (6-10%) were observed. dAMP was incorporated predominantly opposite tetrahydrofuranyl sites positioned in the single strand region of a gapped duplex vector. We conclude from these studies that abasic sites positioned in a bistrand configuration are highly mutagenic in E.coli and COS cells. Repair DNA synthesis may be involved in this process.     

Nucleotide insertion kinetics opposite abasic lesions in DNA

A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.     

Inefficient bypass of an abasic site by DNA polymerase eta

DNA polymerase eta (Pol eta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Pol eta bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O(6)-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Pol eta, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Pol eta are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Pol eta bypasses this lesion extremely poorly. These results suggest that Pol eta requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation.     

Rational attempts to optimize non-natural nucleotides for selective incorporation opposite an abasic site

Translesion DNA synthesis represents the ability of a DNA polymerase to misinsert a nucleotide opposite a DNA lesion. Previous kinetic studies of the bacteriophage T4 DNA polymerase using a series of non-natural nucleotides suggest that pi-electron density of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site, a nontemplating DNA lesion. However, it is surprising that these nonhydrogen-bonding analogues can also be incorporated opposite natural templating DNA with variable degrees of efficiency. In this article, we describe attempts to achieve selectivity for incorporation opposite the abasic site through optimization of pi-electron density and shape of the nucleobase. Toward this goal, we report the synthesis and enzymatic characterization of two novel nucleotide analogues, 5-napthyl-indolyl-2'-deoxyriboside triphosphate (5-NapITP) and 5-anthracene-indolyl-2'-deoxyriboside triphosphate (5-AnITP). The overall catalytic efficiency for their incorporation opposite an abasic site is similar to that of other non-natural nucleotides such as 5-NITP and 5-PhITP that contain significant pi-electron density. As expected, the incorporation of either 5-NapITP or 5-AnITP opposite templating DNA is reduced and presumably reflects steric constraints imposed by the large size of the polycyclic aromatic moieties. Furthermore, 5-NapITP is a chain terminator of translesion DNA synthesis because the DNA polymerase is unable to extend beyond the incorporated non-natural nucleotide. In addition, idle turnover measurements confirm that 5-NapIMP is stably incorporated opposite damaged DNA, and this enhancement reflects the overall favorable incorporation kinetic parameters coupled with a reduction in excision by the exonuclease-proofreading activity of the enzyme. On the basis of these data, we provide a comprehensive assessment of the potential role of pi-electron surface area for nucleotide incorporation opposite templating and nontemplating DNA catalyzed by the bacteriophage T4 DNA polymerase.