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.     

Extrusion of an imperfect palindrome to a cruciform in superhelical DNA: complete determination of energetics using a statistical mechanical model

We present a detailed study of the extrusion of an imperfect palindrome, derived from the terminal regions of vaccinia virus DNA and contained in a superhelical plasmid, into a cruciform containing bulged bases. We monitor the course of extrusion by two-dimensional gel electrophoresis experiments as a function of temperature and linking number. We find that extrusion pauses at partially extruded states as negative superhelicity increases. To understand the course of extrusion with changes in linking number, DeltaLk, we present a rigorous semiempirical statistical mechanical analysis that includes complete coupling between DeltaLk, cruciform extrusion, formation of extrahelical bases, and temperature-dependent denaturation. The imperfections in the palindrome are sequentially incorporated into the cruciform arms as hairpin loops, single unpaired bases, and complex local regions containing several unpaired bases. We analyze the results to determine the free energies, enthalpies and entropies of formation of all local structures involved in extrusion. We find that, for each unpaired structure, the DeltaG, DeltaH and DeltaS of formation are all approximately proportional to the number of unpaired bases contained therein. This surprising result holds regardless of the arrangement or composition of unpaired bases within a particular structure. Imperfections have major effects on the overall energetics of cruciform extrusion and on the course of this transition. In particular, the extent of the DeltaLk change necessary to extrude an imperfect palindrome is considerably greater than that required for extrusion of the underlying perfect palindrome. Our analysis also suggests that, at higher temperatures, significant denaturation at the base of an imperfect cruciform can successfully compete with extension of the cruciform arms.     

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.     

New insights into the structure of abasic DNA from molecular dynamics simulations

Abasic (AP) sites constitute a common form of DNA damage, arising from the spontaneous or enzymatic breakage of the N-glycosyl bond and the loss of a nucleotide base. To examine the effects of such damage on DNA structure, especially in the vicinity of the abasic sugar, four 1.5 ns molecular dynamics simulations of double-helical DNA dodecamers with and without a single abasic (tetrahydrofuran, X) lesion in a 5′-d(CXT) context have been performed and analyzed. The results indicate that the abasic site does not maintain a hole or gap in the DNA, but instead perturbs the canonical structure and induces additional flexibility close to the abasic site. In the apurinic simulations (i.e., when a pyrimidine is opposite the AP site), the abasic sugar flipped in and out of the minor groove, and the gap was water filled, except during the occurrence of a novel non-Watson–Crick C-T base pair across the abasic site. The apyrimidinic gap was not penetrated by water until the abasic sugar flipped out and remained extrahelical. Both AP helices showed kinks of 20–30° at the abasic site. The Watson–Crick hydrogen bonds are more transient throughout the DNA double helices containing an abasic site. The abasic sugar displayed an unusually broad range of sugar puckers centered around the northern pucker. The increased motion of the bases and backbone near the abasic site appear to correlate with sequence-dependent helical stability. The data indicate that abasic DNA contorts more easily and in specific ways relative to unmodified DNA, an aspect likely to be important in abasic site recognition and hydrolysis.     

The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

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.     

The Fen1 extrahelical 3'-flap pocket is conserved from archaea to human and regulates DNA substrate specificity

Fen1 is a key enzyme for the maintenance of genetic stability in archaea and eukaryotes and is classified as a tumor suppressor. Very recent structural data obtained from Archaeoglobus fulgidus Fen1 suggest that an extrahelical 3'-flap pocket is responsible for substrate specificity, by binding to the unpaired 3'-flap and by opening and kinking the DNA. Since the extrahelical 3'-flap pocket in archaeal Fen1 contains seven amino acids that are conserved to a great extent in human Fen1, we have mutated the four conserved or all seven amino acids in the human Fen1 extrahelical 3'-flap pocket to alanine. Our data suggest that the human extrahelical 3'-flap pocket mutants have lost substrate specificity to the double-flap DNA. Moreover, loss of high affinity for the unpaired 3'-flap suggests that the extrahelical 3'-flap pocket is essential for recognition and processing of the 'physiological' template. Human PCNA could stimulate the human Fen1 extrahelical 3'-flap pocket mutants but not restore their specificity. Thus the substrate specificity of Fen1 has been functionally conserved over a billion years from archaea to human.     

Individual nucleotide bases, not base pairs, are critical for triggering site-specific DNA cleavage by vaccinia topoisomerase

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of abasic lesions at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. The rate of DNA incision was reduced by factors of 350, 250, 60, and 10 when abasic sites replaced the -1N, +1T, +2T, and +4C bases of the scissile strand, but abasic lesions at +5C and +3C had little or no effect. Abasic lesions in the nonscissile strand in lieu of +4G, +3G, +2A, and +1A reduced the rate of cleavage by factors of 130, 150, 10, and 5, whereas abasic lesions at +5G and -1N had no effect. The striking positional asymmetry of abasic interference on the scissile and nonscissile strands highlights the importance of individual bases, not base pairs, in promoting DNA cleavage. The rate of single-turnover DNA religation by the covalent topoisomerase-DNA complex was insensitive to abasic sites within the CCCTT sequence of the scissile strand, but an abasic lesion at the 5'-OH nucleoside (-1N) of the attacking DNA strand slowed the rate of religation by a factor of 600. Nonscissile strand abasic lesions at +1A and -1N slowed the rate of religation by factors of approximately 140 and 20, respectively, and strongly skewed the cleavage-religation equilibrium toward the covalent complex. Thus, abasic lesions immediately flanking the cleavage site act as topoisomerase poisons.     

Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine

Abasic sites are highly mutagenic lesions in DNA that arise as intermediates in the excision repair of modified bases. These sites are generated by the action of damage-specific DNA glycosylases and are converted into downstream intermediates by the specific activity of apurinic/apyrimidinic endonucleases. Enzymes in both families have been observed in crystal structures to impose deformations on the abasic-site DNA, including DNA kinking and base flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contribute to the specificity of these repair enzymes. Previously, measurements of the steady-state fluorescence of the adenine analogue 2-aminopurine (2AP) opposite an abasic site demonstrated that binding of divalent cations could induce a conformational change that increased the accessibility of 2AP to solute quenching [Stivers, J. T. (1998) Nucleic Acids Res. 26, 3837-44]. We have performed time-resolved fluorescence experiments to characterize the states involved in this conformational change. Interpretation of these studies is based on a recently developed model attributing the static and dynamic fluorescence quenching of 2AP in DNA to aromatic stacking and collisional interactions with neighboring bases, respectively (see the preceding paper in this issue). The time-resolved fluorescence results indicate that divalent cation binding shifts the equilibrium of the abasic site between two conformations: a "closed" state, characterized by short average fluorescence lifetime and complex decay kinetics, and an "open" state, characterized by monoexponential decay with lifetime approximately that of the free nucleoside. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neighboring bases, the absence of dynamic quenching in the open state strongly suggests that the fluorescent base is extrahelical in this conformation. Consistent with this interpretation, time-resolved quenching studies reveal that the open state is accessible to solute quenching by potassium iodide, but the closed state is not. Greater static quenching is observed in the abasic site when the fluorescent base is flanked by 5'- and 3'-thymines than in the context of 5'- and 3'-adenines, indicating that 2AP is more stacked with the neighboring bases in the former sequence. These results imply that the conformation of the abasic site varies in a sequence-dependent manner. Undamaged sequences in which the abasic site is replaced by thymine do not exhibit an open state and have different levels of both static and dynamic quenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of repair enzymes for the abasic site.     

Activities and incision patterns of ABC excinuclease on modified DNA containing single-base mismatches and extrahelical bases

ABC excision nuclease of Escherichia coli is a DNA repair enzyme that recognizes major helical distortions caused by bulky base adducts and incises on both sides of the adduct, thus removing the modified nucleotides in the form of a 12-13-base long oligomer. We tested the enzyme with substrates that contained unusual helical structures caused by single-base mismatches or one, three, or four extrahelical bases (loops). We find that the enzyme does not cut DNAs containing helical perturbations caused by these structures. However, when the mismatched or extrahelical bases are modified with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide, a reagent specific for unpaired G and T residues, the enzyme incises at the modified nucleotides in the regular manner. In addition, we find that when mismatches and loops are located near pyrimidine dimers and (6-4) photoproducts they do not inhibit incision at the photoproducts by the excinuclease but sometimes affect the incision pattern. Our results indicate that ABC excinuclease may be a useful enzymatic reagent to probe the structural changes caused by mismatches and deletions in DNA and provide additional information on the requirements for incision by this repair enzyme.     

NMR studies of a deoxyribodecanucleotide containing an extrahelical thymidine surrounded by an oligo(dA).oligo(dT) tract

One- and two-dimensional NMR experiments were carried out on a decamer, d-(CGCTTTTCGC).d(GCGAAAAGCG), and on the same sequence with the addition of an unpaired thymidine, d(CGCTTTTCGC).d(GCGAATAAGCG), which will be referred to as the T-bulge decamer. Evidence from one-dimensional NOE experiments on the exchangeable protons indicates that the unpaired thymidine is extrahelical. This conclusion is also supported by numerous cross-peaks in the two-dimensional NOESY spectrum of the nonexchangeable protons. Assignments for all of the resonances, with the exception of the H5' and H5" resonances, have been made for both oligonucleotide duplexes through the use of 2D NOESY, COSY, and relayed COSY experiments. Temperature dependence of the methyl resonance chemical shifts indicates that the unpaired thymidine shows unusual behavior compared to other thymidines in the duplex. Two-dimensional NOESY experiments carried out from 5 to 35 degrees C indicate the unpaired thymidine remains extrahelical throughout this temperature range. A similar temperature dependence for the methyl chemical shift is found in the corresponding single-strand d(GCGAATAAGCG). The oligo-(dA).oligo(dT) tracts in both the decamer and the T-bulge decamer have structures different from B-form DNA and exhibit NOEs similar to those observed in other oligonucleotides containing A.T tracts. The formation of this unusual A.T tract structure may induce the extrahelical conformation of the unpaired thymidine.     

Induction of double-strand breaks by S1 nuclease, mung bean nuclease and nuclease P1 in DNA containing abasic sites and nicks.

Defined DNA substrates containing discrete abasic sites or paired abasic sites set 1, 3, 5 and 7 bases apart on opposite strands were constructed to examine the reactivity of S1, mung bean and P1 nucleases towards abasic sites. None of the enzymes acted on the substrate containing discrete abasic sites. Under conditions where little or no non-specific DNA degradation was observed, all three nucleases were able to generate double-strand breaks when the bistranded abasic sites were 1 and 3 base pairs apart. However, when the abasic sites were further apart, the enzymes again failed to cleave the DNA. These results indicate that single abasic sites do not cause sufficient denaturation of the DNA to allow incision by these single-strand specific endonucleases. The reactivity of these enzymes was also investigated on DNA substrates that were nicked by DNasel or more site-specifically by endonuclease III incision at the discrete abasic sites. The three nucleases readily induced a strand break opposite such nicks.     

Correlation of bistranded clustered abasic DNA lesion processing with structural and dynamic DNA helix distortion

Clustered apurinic/apyrimidinic (AP; abasic) DNA lesions produced by ionizing radiation are by far more cytotoxic than isolated AP lesion entities. The structure and dynamics of a series of seven 23-bp oligonucleotides featuring simple bistranded clustered damage sites, comprising of two AP sites, zero, one, three or five bases 3′ or 5′ apart from each other, were investigated through 400 ns explicit solvent molecular dynamics simulations. They provide representative structures of synthetically engineered multiply damage sites-containing oligonucleotides whose repair was investigated experimentally (Nucl. Acids Res. 2004, 32:5609-5620; Nucl. Acids Res. 2002, 30: 2800–2808). The inspection of extrahelical positioning of the AP sites, bulge and non Watson–Crick hydrogen bonding corroborates the experimental measurements of repair efficiencies by bacterial or human AP endonucleases Nfo and APE1, respectively. This study provides unprecedented knowledge into the structure and dynamics of clustered abasic DNA lesions, notably rationalizing the non-symmetry with respect to 3′ to 5′ position. In addition, it provides strong mechanistic insights and basis for future studies on the effects of clustered DNA damage on the recognition and processing of these lesions by bacterial or human DNA repair enzymes specialized in the processing of such lesions.     

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.     

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.     

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.     

Snapshots of replication through an abasic lesion; structural basis for base substitutions and frameshifts

Dpo4 from S. Solfataricus, a DinB-like Y family polymerase, efficiently replicates DNA past an abasic lesion. We have determined crystal structures of Dpo4 complexed with five different abasic site-containing DNA substrates and find that translesion synthesis is template directed with the abasic site looped out and the incoming nucleotide is opposite the base 5' to the lesion. The ensuing DNA synthesis generates a -1 frameshift when the abasic site remains extrahelical. Template realignment during primer extension is also observed, resulting in base substitutions or even +1 frameshifts. In the case of a +1 frameshift, the extra nucleotide is accommodated in the solvent-exposed minor groove. In addition, the structure of an unproductive Dpo4 ternary complex suggests that the flexible little finger domain facilitates DNA orientation and translocation during translesion synthesis.     

The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence

Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA–protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition.