Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase iota

The 1,N6-ethenodeoxyadenosine (epsilon dA) lesion is promutagenic and has been implicated in carcinogenesis. We show here that human Pol iota, a Y-family DNA polymerase, can promote replication through this lesion by proficiently incorporating a nucleotide opposite it. The structural basis of this action is rotation of the epsilon dA adduct to the syn conformation in the Pol iota active site and presentation of its 'Hoogsteen edge' for hydrogen-bonding with incoming dTTP or dCTP. We also show that Pol zeta carries out the subsequent extension reaction and that efficiency of extension from epsilon dA x T is notably higher than from epsilon dA x C. Together, our studies reveal for the first time how the exocyclic epsilon dA adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Pol iota and Pol zeta provides for efficient and error-free synthesis through this potentially carcinogenic DNA lesion.     

Preferential incorporation of G opposite template T by the low-fidelity human DNA polymerase iota

DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Pol(iota), encoded by the RAD30B gene. We show that human Pol(iota) violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Pol(iota) preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Pol(iota) compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Pol(iota) to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Pol(iota) copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Pol(iota) incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Pol(iota) in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Pol(iota) may additionally play a specialized function in human biology.     

Human DNA polymerase iota utilizes different nucleotide incorporation mechanisms dependent upon the template base

Human DNA polymerase ι (Polι) is a member of the Y family of DNA polymerases involved in translesion DNA synthesis. Polι is highly unusual in that it possesses a high fidelity on template A, but has an unprecedented low fidelity on template T, preferring to misincorporate a G instead of an A. To understand the mechanisms of nucleotide incorporation opposite different template bases by Polι, we have carried out pre-steady-state kinetic analyses of nucleotide incorporation opposite templates A and T. These analyses have revealed that opposite template A, the correct nucleotide is preferred because it is bound tighter and is incorporated faster than the incorrect nucleotides. Opposite template T, however, the correct and incorrect nucleotides are incorporated at very similar rates, and interestingly, the greater efficiency of G misincorporation relative to A incorporation opposite T arises predominantly from the tighter binding of G. Based on these results, we propose that the incipient base pair is accommodated differently in the active site of Polι dependent upon the template base and that when T is the templating base, Polι accommodates the wobble base pair better than the Watson-Crick base pair.     

Alternative heterocycles for DNA recognition: a 3-pyrazole/pyrrole pair specifies for G.C base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5'-ATNCCTAA-3' (N = G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G.C bp compared with Im/Py.     

Human DNA polymerase iota promotes replication through a ring-closed minor-groove adduct that adopts a syn conformation in DNA

Acrolein, an alpha,beta-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from oxidation of polyamines. The reaction of acrolein with the N2 group of guanine in DNA leads to the formation of a cyclic adduct, gamma-hydroxy-1,N2-propano-2'-deoxyguanosine (gamma-HOPdG). Previously, we have shown that proficient replication through the gamma-HOPdG adduct can be mediated by the sequential action of human DNA polymerases (Pols) iota and kappa, in which Poliota incorporates either pyrimidine opposite gamma-HOPdG, but Polkappa extends only from the cytosine. Since gamma-HOPdG can adopt either a ring-closed cyclic form or a ring-opened form in DNA, to better understand the mechanisms that Pols iota and kappa employ to promote replication through this lesion, we have examined the ability of these polymerases to replicate through the structural analogs of gamma-HOPdG that are permanently either ring closed or ring opened. Our studies with these model adducts show that whereas the ring-opened form of gamma-HOPdG is not inhibitory to synthesis by human Pols eta, iota, or kappa, only Poliota is able to incorporate nucleotides opposite the ring-closed form, which is known to adopt a syn conformation in DNA. From these studies, we infer that (i) Pols eta, iota, and kappa have the ability to proficiently replicate through minor-groove DNA lesions that do not perturb the Watson-Crick hydrogen bonding of the template base with the incoming nucleotide, and (ii) Poliota can accommodate a minor-groove-adducted template purine which adopts a syn conformation in DNA and forms a Hoogsteen base pair with the incoming nucleotide.     

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

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.     

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.     

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.     

Construction of a 42 base pair double stranded DNA microcircle.

A procedure for the construction of double stranded DNA microcircles is described that overcomes the natural limits of established circularization procedures. Starting with two synthetic oligonucleotides which are able to form dumbbell shaped structures, two subsequent ligation reactions yield a microcircle of double stranded DNA of 42 base pairs. This is by far the smallest circle of double stranded DNA yet described. These microcircles can be constructed in quantities required for high resolution structural analyses such as X-ray crystallography and NMR spectroscopy.     

Photochemical demonstration of stacked C.C+ base pairs in a novel DNA secondary structure

The secondary structure of the alternating polydeoxynucleotide sequence poly[d(C-T)] was studied as a function of pH by ultraviolet absorbance and circular dichroism spectroscopy and by the analysis of UV-induced photoproducts. As the pH was lowered, poly[d(C-T)] underwent a conformational transition that was characterized by changes in the long-wavelength region (280-320 nm) of the CD spectrum. These changes have previously been interpreted as evidence for the formation of a core of stacked, protonated C X C+ base pairs in a double-helical complex of poly[d(C-T)], with the thymidyl residues being looped out into the solvent [Gray, D. M., Vaughan, M., Ratliff, R. L., & Hayes, F. N. (1980) Nucleic Acids Res. 8, 3695-3707]. In the present work, poly[d(C-T)] was labeled with [U-14C]cytosine and [methyl-3H]thymine and irradiated at pH values both above and below the conformational transition point (monitored by CD spectroscopy). The distribution of radioactivity in uracil means value of uracil dimers, uracil means value of thymine dimers (the deamination products of cytosine means value of cytosine and cytosine means value of thymine dimers, respectively), and thymine-means value of thymine dimers was then determined. As the pH was decreased, we found an increase in the yield of uracil means value of uracil dimers and a decrease in the yield of uracil means value of thymine dimers, which occurred concomitantly with the change in the CD spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

A.T and C.C+ base pairs can form simultaneously in a novel multistranded DNA complex

Previous experiments have established that in certain synthetic oligomeric DNA sequences, including mixtures of d(AACC)5 with d(CCTT)5, adenine-thymine (A.T) base pairs form to the exclusion of neighboring protonated cytosine-cytosine (C.C+) base pairs [Edwards, E., Ratliff, R., & Gray, D. (1988) Biochemistry 27, 5166-5174]. In the present work, circular dichroism and other measurements were used to study DNA oligomers that represented two additional classes with respect to the formation of A.T and/or C.C+ base pairs. (1) One class included two sets of repeating pentameric DNA sequences, d(CCAAT)3-6 and d(AATCC)4,5. For both of these sets of oligomers, an increase in the magnitude of the long-wavelength positive CD band centered at about 280 nm occurred as the pH was lowered from 7 to 5 at 0.1 and 0.5 M Na+, indicating that C.C+ base pairs formed. Even though it may have been possible for these oligomers to form duplexes with two antiparallel A.T base pairs per pentamer, no A.T base pairing was detected by monitoring the CD changes at 250 nm. Thus, spectral data showed that as few as 40% C.C+ base pairs were stable in two sets of oligomers in which A.T base pairs did not form adjacent to, or in place of, C.C+ base pairs. (2) Another class of oligomer was represented by d(C4A4T4C4), which was studied by CD, HPLC, and centrifugation experiments. We confirmed previous work that this sequence was able to form both types of base pairs as the pH and temperature were lowered [Gray, D., Cui, T., & Ratliff, R. (1984) Nucleic Acids Res. 12, 7565-7580].(ABSTRACT TRUNCATED AT 250 WORDS)     

The post-incision steps of the DNA base excision repair pathway in Escherichia coli: studies with a closed circular DNA substrate containing a single U:G base pair.

The DNA base excision repair pathway is responsible for removal of oxidative and endogenous DNA base damage in both prokaryotes and eukaryotes. This pathway involves formation of an apurinic/apyrimidinic (AP) site in the DNA, which is further processed to restore the integrity of the DNA. In Escherichia coli it has been suggested that the major mode of repair involves replacement of a single nucleotide at the AP site, based on repair synthesis studies using oligonucleotide substrates containing a unique uracil base. The mechanism of the post-incision steps of the bacterial base excision repair pathway was examined using a DNA plasmid substrate containing a single U:G base pair. Repair synthesis carried out by repair-proficient ung, recJ and xon E.coli cell extracts was analyzed by restriction endonuclease cleavage of the DNA containing the uracil lesion. It was found that replacement of the uracil base was always accompanied by replacement of several nucleotides ( approximately 15) 3' of the uracil and this process was absolutely dependent on initial removal of the uracil base by the action of uracil-DNA glycosylase. In contrast to findings with oligonucleotide substrates, replacement of just a single nucleotide at the lesion site was not detected. These results suggest that repair patch length may be substrate dependent and a re-evaluation of the post-incision steps of base excision repair is suggested.     

Increased G + C content of DNA stabilizes methyl CpG dinucleotides.

The vertebrate genome is a mosaic of regions differing dramatically in their G + C content. Those regions with a high G + C content contain the expected number of CpG dinucleotides and we propose that following methylation these have been protected from deamination by the increased stability of the surrounding DNA duplex. This argument applies both to the microenvironment of the CpG dinucleotide and to whole gene regions.     

Evidence for Watson-Crick and not Hoogsteen or wobble base pairing in the selection of nucleotides for insertion opposite pyrimidines and a thymine dimer by yeast DNA pol eta

We have recently reported that pyrene nucleotide is preferentially inserted opposite an abasic site, the 3'-T of a thymine dimer, and most undamaged bases by yeast DNA polymerase eta (pol eta). Because pyrene is a nonpolar molecule with no H-bonding ability, the unusually high efficiencies of dPMP insertion are ascribed to its superior base stacking ability, and underscore the importance of base stacking in the selection of nucleotides by pol eta. To investigate the role of H-bonding and base pair geometry in the selection of nucleotides by pol eta, we determined the insertion efficiencies of the base-modified nucleotides 2,6-diaminopurine, 2-aminopurine, 6-chloropurine, and inosine which would make a different number of H-bonds with the template base depending on base pair geometry. Watson-Crick base pairing appears to play an important role in the selection of nucleotide analogues for insertion opposite C and T as evidenced by the decrease in the relative insertion efficiencies with a decrease in the number of Watson-Crick H-bonds and an increase in the number of donor-donor and acceptor-acceptor interactions. The selectivity of nucleotide insertion is greater opposite the 5'-T than the 3'-T of the thymine dimer, in accord with previous work suggesting that the 5'-T is held more rigidly than the 3'-T. Furthermore, insertion of A opposite both Ts of the dimer appears to be mediated by Watson-Crick base pairing and not by Hoogsteen base pairing based on the almost identical insertion efficiencies of A and 7-deaza-A, the latter of which lacks H-bonding capability at N7. The relative efficiencies for insertion of nucleotides that can form Watson-Crick base pairs parallel those for the Klenow fragment, whereas the Klenow fragment more strongly discriminates against mismatches, in accord with its greater shape selectivity. These results underscore the importance of H-bonding and Watson-Crick base pair geometry in the selection of nucleotides by both pol eta and the Klenow fragment, and the lesser role of shape selection in insertion by pol eta due to its more open and less constrained active site.     

Imidazole-imidazole pair as a minor groove recognition motif for T:G mismatched base pairs.

The T:G mismatched base pair is associated with many genetic mutations. Understanding its biological consequences may be aided by studying the structural perturbation of DNA caused by a T:G base pair and by specific probing of the mismatch using small molecular ligands. We have shown previously that AR-1-144, a tri-imidazole (Im-Im-Im) minor groove binder, recognizes the sequence CCGG. NMR structural analysis of the symmetric 2:1 complex of AR-1-144 and GAACCGGTTC revealed that each AR-1-144 binds to four base pairs with the guanine N2 amino group forming a bifurcated hydrogen bond to a side-by-side Im/Im pair. We predicted that the free G-N2 amino group in a T:G wobble base pair can form two individual hydrogen bonds to a side-by-side Im/Im pair. Thus an Im/Im pair may be a good recognition motif for a T:G base pair in DNA. Cooperative and tight binding of an AR-1-144 homodimer to GAACTGGTTC permits a detailed structural analysis by 2D NOE NMR refinement and the refined structure confirms our prediction. Surprisingly, AR-1-144 does not bind to GAATCGGTTC. We further show that both the Im-Im-Im/Im-Py-Im heterodimer and the Im-Im-Im/Im-Im-Im homodimer bind strongly to the CACGGGTC + GACTCGTG duplex. These results together suggest that an Im/Im pair can specifically recognize a single T:G mismatch. Our results may be useful in future design of molecules (e.g. linked dimers) that can recognize a single T:G mismatch with specificity.