Hoogsteen Research Articles

Synthesis of C‐Nucleosides Designed to Participate in Triplex Formation with Native DNA: Specific Recognition of an A:T Base Pair in DNA.

We have previously described a system of 2-aminoquinoline- and 2-aminoquinazoline-based C-deoxynucleosides (TRIPsides) that are designed to be incorporated into oligomers that can specifically bind in the major groove via Hoogsteen base pairing to any sequence of native DNA. The four TRIPsides are termed antiGC, antiCG, antiTA, and antiAT with respect to the Watson−Crick base pair targets that they bind. The first three TRIPsides have been prepared, characterized, and shown to form stable and sequence-specific triplexes. In the present study, we describe the preparation of two molecules, 2-amino-4-(2‘-deoxy-β-d-ribofuranosyl)quinazoline (7) and 2-amino-6-fluoro-4-(2‘-deoxy-β-d-ribofuranosyl)quinoline (14), that can serve as the remaining antiAT TRIPside. The phosphoramidites of 7 and 14 were prepared, but only the latter was successfully incorporated into DNA oligomers. It is demonstrated using UV−visible melting experiments that 14 forms sequence-specific intramolecular triplets with A:T base pairs at ph...

Replication past a trans-4-Hydroxynonenal Minor-Groove Adduct by the Sequential Action of Human DNA Polymerases ι and κ

The X-ray crystal structure of human DNA polymerase iota (Poliota) has shown that it differs from all known Pols in its dependence upon Hoogsteen base pairing for synthesizing DNA. Hoogsteen base pairing provides an elegant mechanism for synthesizing DNA opposite minor-groove adducts that present a severe block to synthesis by replicative DNA polymerases. Germane to this problem, a variety of DNA adducts form at the N2 minor-groove position of guanine. Previously, we have shown that proficient and error-free replication through the gamma-HOPdG (gamma-hydroxy-1,N2-propano-2'-deoxyguanosine) adduct, which is formed from the reaction of acrolein with the N2 of guanine, is mediated by the sequential action of human Poliota and Polkappa, in which Poliota incorporates the nucleotide opposite the lesion site and Polkappa carries out the subsequent extension reaction. To test the general applicability of these observations to other adducts formed at the N2 position of guanine, here we examine the proficiency of human Poliota and Polkappa to synthesize past stereoisomers of trans-4-hydroxy-2-nonenal-deoxyguanosine (HNE-dG). Even though HNE- and acrolein-modified dGs share common structural features, due to their increased size and other structural differences, HNE adducts are potentially more blocking for replication than gamma-HOPdG. We show here that the sequential action of Poliota and Polkappa promotes efficient and error-free synthesis through the HNE-dG adducts, in which Poliota incorporates the nucleotide opposite the lesion site and Polkappa performs the extension reaction.

Efficient and High Fidelity Incorporation of dCTP Opposite 7,8-Dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA Polymerase Dpo4

DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed >/=95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured K(d)(,dCTP) was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The E(a) for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca(2+). The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.

A Lysine Residue in the Fingers Subdomain of T7 DNA Polymerase Modulates the Miscoding Potential of 8-Oxo-7,8-Dihydroguanosine

8-oxo-7,8-dihydroguanosine (8oG) is a highly mutagenic DNA lesion that stably pairs with adenosine, forming 8oG(syn).dA(anti) Hoogsteen base pairs. DNA polymerases show different propensities to insert dCMP or dAMP opposite 8oG, but the molecular mechanisms that determine faithful or mutagenic bypass are poorly understood. Here, we report kinetic and structural data providing evidence that, in T7 DNA polymerase, residue Lys536 is responsible for attenuating the miscoding potential of 8oG. The Lys536Ala polymerase shows a significant increase in mutagenic 8oG bypass versus wild-type polymerase, and a crystal structure of the Lys536Ala mutant reveals a closed complex with an 8oG(syn).dATP mismatch in the polymerase active site, in contrast to the unproductive, open complex previously obtained by using wild-type polymerase. We propose that Lys536 acts as a steric and/or electrostatic filter that attenuates the miscoding potential of 8oG by normally interfering with the binding of 8oG in a syn conformation that pairs with dATP.

Human DNA Polymerase ι Incorporates dCTP Opposite Template G via a G.C+ Hoogsteen Base Pair

Human DNA polymerase iota (hPoliota), a member of the Y family of DNA polymerases, differs in remarkable ways from other DNA polymerases, incorporating correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. We present here the crystal structure of hPoliota bound to template G and incoming dCTP, which reveals a G.C + Hoogsteen base pair in a DNA polymerase active site. We show that the hPoliota active site has evolved to favor Hoogsteen base pairing, wherein the template sugar is fixed in a cavity that reduces the C1'-C1' distance across the nascent base pair from approximately 10.5 A in other DNA polymerases to 8.6 A in hPoliota. The rotation of G from anti to syn is then largely in response to this curtailed C1'-C1' distance. A G.C+ Hoogsteen base pair suggests a specific mechanism for hPoliota's ability to bypass N(2)-adducted guanines that obstruct replication.

Human DNA Polymerase ι 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.

Synthesis of C-Nucleosides Designed To Participate in Triplex Formation with Native DNA: Specific Recognition of an A:T Base Pair in DNA

[structure: see text] We have previously described a system of 2-aminoquinoline- and 2-aminoquinazoline-based C-deoxynucleosides (TRIPsides) that are designed to be incorporated into oligomers that can specifically bind in the major groove via Hoogsteen base pairing to any sequence of native DNA. The four TRIPsides are termed antiGC, antiCG, antiTA, and antiAT with respect to the Watson-Crick base pair targets that they bind. The first three TRIPsides have been prepared, characterized, and shown to form stable and sequence-specific triplexes. In the present study, we describe the preparation of two molecules, 2-amino-4-(2'-deoxy-beta-D-ribofuranosyl)quinazoline (7) and 2-amino-6-fluoro-4-(2'-deoxy-beta-D-ribofuranosyl)quinoline (14), that can serve as the remaining antiAT TRIPside. The phosphoramidites of 7 and 14 were prepared, but only the latter was successfully incorporated into DNA oligomers. It is demonstrated using UV-visible melting experiments that 14 forms sequence-specific intramolecular triplets with A:T base pairs at physiological pH.

Hoogsteen base-pairing in DNA replication?

Human polymerase-iota belongs to the error-prone Y family of polymerases, which frequently incorporate incorrect nucleotides during DNA replication but can efficiently bypass DNA lesions. On the basis of X-ray diffraction data, Nair et al. propose that Hoogsteen base-pairing is adopted by DNA during its replication by this enzyme. Here I re-examine their X-ray data and find that the electron density is very weak for a Hoogsteen base pair formed between a template adenine deoxyribonucleotide in the syn conformation and a deoxythymidine 5'-triphosphate (dTTP), and that the fit is better for a normal Watson-Crick base pair. As a guanine-cytosine (G-C) base pair has no potential to form a Hoogsteen base pair at physiological pH, Hoogsteen base-pairing is unlikely to be used in replication by this polymerase.

Hoogsteen base-pairing in DNA replication? (reply)

We have solved the crystal structure of human DNA polymerase-ι (hPolι) bound to a template primer and incoming nucleotide, and shown that hPolι uses Hoogsteen base-pairing for normal DNA synthesis1. Jimin Wang questions the space group we used and claims that Hoogsteen base-pairing is not supported by our electron-density maps2. We show here that the issue of space group does not affect our conclusions1 and that our electron-density maps are clear in indicating that Hoogsteen and not Watson–Crick base-pairing occurs in this situation.

Serendipitous SAD phasing

We describe here the solution of the structure of a bisintercalatorDNA complex with the help of an anomalous scatterer fortuitously present in the structure. The DNA bisintercalator Echinomycin is characteristic of a family of anticancer drugs that interfere with both replication and transcription. The crystal structures of Triostin A[1] and Echinomycin[1] in complex with (CGTACG)2 showed for the first time Hoogsteen base pairing in a double stranded DNA. In our laboratory we grew crystals of the complex of Echinomycin with two other sequences: (GCGTACGC)2 and (ACGTACGT)2, which show also the canonical Hoogsteen base pairing outside the intercalation site. Over time, a different crystal form grew in the the same drops whose structure could not be solved by molecular replacement. A native high resolution dataset was collected at BL14.1 at BESSY on this crystal form and showed a weak but significant anomalous signal although no heavy atom was included in the crystallization conditions. SAD phasing showed one single heavy atom site from which an interpretable electron density map could be obtained. The heavy atom proved to be a mixture of metals of unknown origin. The structure shows a mixture of the canonical Hoogsteen base pairing together with the Watson-Crick mode that was suggested by NMR data, showing great flexibility of the DNA duplex in accommodating the bisintercalation and that Hoogsteen base pairing is not a necessary condition for Echinomycin binding. It offers a detailed three-dimensional model for molecular dynamics simulations of binding for both Hoogsteen and Watson-Crick base pairing around the intercalation site.

Distinct mechanisms of cis-syn thymine dimer bypass by Dpo4 and DNA polymerase η

UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Poleta promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Poleta, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Poleta. The x-ray crystal structure of Dpo4 complexed with either the 3'-thymine (T) or the 5' T of a cis-syn TT dimer has indicated that, whereas the 3' T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5' T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5' T of the dimer with the incoming A has been proposed for Poleta. Here we examine the mechanisms of CPD bypass by Dpo4 and Poleta using nucleotide analogs that specifically disrupt the Hoogsteen or Watson-Crick base pairing. Our results show that both Dpo4 and Poleta incorporate dATP opposite the 5' T of the CPD via Watson-Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3' T of the dimer, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Poleta using the normal Watson-Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols.

Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro

The POT1 (protection of telomeres 1) protein binds the ssDNA overhangs at the ends of chromosomes in diverse eukaryotes. POT1 is essential for chromosome end-protection, as best demonstrated in fission yeast. In human cells, hPOT1 is also involved in telomere-length regulation. We now show that telomeric oligonucleotides, such as d[GGG(TTAGGG)(3)], which form intramolecular G-quadruplexes through Hoogsteen base-pairing, serve as only marginal primers for extension by recombinant human telomerase; telomerase stalls after every nucleotide addition. Addition of hPOT1 to the reaction restores the normal processive elongation pattern seen with primers that cannot form G-quadruplexes. hPOT1 does not act catalytically but, instead, forms a stoichiometric complex with the DNA, freeing its 3' tail. An antisense oligonucleotide, which base-pairs near the 5' end of the telomeric sequence, leaving a telomerase-extendable 3' tail, duplicates the effect of hPOT1 on activation of G-quadruplex primers. Thus, hPOT1 may function simply by trapping the unfolded forms of these telomeric primers in an equilibrium population. We propose an additional role for hPOT1 in telomere maintenance: disrupting G-quadruplex structures in telomeric DNA, thereby allowing proper elongation by telomerase.

Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase ι

Because of the near geometric identity of Watson-Crick (W-C) GxC and AxT base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase iota (Pol iota), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Pol iota is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Pol iota active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Pol iota ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Pol iota.

Highly Stable DNA Triplexes Formed with Cationic Phosphoramidate Pyrimidine α‐Oligonucleotides

The ability of cationic phosphoramidate pyrimidine alpha-oligonucleotides (ONs) to form triplexes with DNA duplexes was investigated by UV melting experiments, circular dichroism spectroscopy and gel mobility shift experiments. Replacement of the phosphodiester linkages in alpha-ONs with positively charged phosphoramidate linkages results in more efficient triplex formation, the triplex stability increasing with the number of positive charges. At a neutral pH and in the absence of magnesium ions, it was found that a fully cationic phosphoramidate alpha-TFO (triplex-forming oligonucleotide) forms a highly stable triplex that melts at a higher temperature than the duplex target. No hysteresis between the annealing and melting curves was noticed; this indicates fast association. Moreover, the recognition of a DNA duplex with a cationic alpha-TFO through Hoogsteen base pairing is highly sequence-specific. To the best of our knowledge, this is the first report of stable triplexes in the pyrimidine motif formed by cationic alpha-oligonucleotides and duplex targets.

Influence of Ions, Hydration, and the Transcriptional Inhibitor P4N on the Conformations of the Sp1 Binding Site

Three crystal structures containing the entire Sp1 consensus sequence d(GGGGCGGGG) with two or three additional base-pairs on either the 5' or 3' ends and overhangs have been determined. Despite the different lengths of DNA in the pseudo-dodecamers and pseudo-tridecamer, all three structures form A-DNA duplexes that share a common set of crystal contacts, including a T*(G.C) base triplet and a 5'-overhang that flips out and away from the helical axes to form a Hoogsteen base-pair with the 3'-overhang of a symmetry mate. The global conformations of the three structures differ, however, in the widths of their respective major grooves, the lengths of the molecules, and the extent of crystal packing. The structures were determined from crystals grown in an unusual precipitant for A-DNA, polyethylene glycol (PEG) 400, in combination with polyamines or ions; cobalt hexamine for the pseudo-tridecamer, and spermidine for the pseudo-dodecamers. As the Sp1 binding site is a target for antiviral and anticancer drugs, pseudo-dodecamer crystals were soaked with one such antiviral and anticancer compound, P4N. Although P4N was not visualized unambiguously in the electron density maps, the effect of the drug is evident from significant differences in the lattice constants, crystal packing, and overall conformation of the structure.

Stabilization of purine motif DNA triplex by a tetrapeptide from the binding domain of HMGBI protein

DNA triple helix has attracted a lot of attention in the recent past because of their potential therapeutic applications to inhibit the transcription of genes associated with several human diseases. Therefore, studies on DNA triplex formation and stabilization have become very essential to understand the nature of DNA triplex both in quantitative and qualitative terms. We have studied the effect of a tetrapeptide, Pro-Lys-Arg-Trp (PKRW), containing the conserved sequence (PKR) of high mobility group (HMG) B1 DNA binding domain on the stability of purine motif DNA triplex. The triple stranded DNA complex (23R*R•Y) containing (GAA) repeats used in the present study is formed by: 5′-TCGCGAAGAAGAAGAAGAACGCT-3′, 5′-AGCGTTCTTCTTCTTCTTCGCGA-3′ and the third strand 5′-AAGAAGAAGAAGAAG-3′. The DNA triplex 23R*R•Y and its complex with PKRW were investigated by DNA-melting, UV-absorption, circular dichroism and fluorescence spectroscopy. The increased stability of 23R*R•Y triplex in presence of PKRW is supported by the following observations; (i) a marked increase in triplex melting temperature (ii) significant increase in the ellipticity of the characteristic negative band at 210 nm and (iii) a large change in free energy, Δ Δ G (Δ G of triplex-peptide complex – Δ G of free triplex), –9.6 kcal mol –1 on complexation with PKRW. Based on the above observations and fluorescence quenching data, we suggest a two stage model for the binding of PKRW to 23R*R•Y in which (a) PKRW binds to the minor groove of the 23R*R•Y with lysine and arginine making electrostatic interactions with phosphates of DNA, which is followed by (b) intercalation of tryptophan side chain between the A*A•T base triplets. We conclude that PKRW preferentially stabilizes Hoogsteen base pair to the Watson–Crick base pair in purine motif 23R*R•Y DNA triplex. It is tempting to speculate that HMGB1 plays an important role in recognizing the DNA triplex structures like H-DNA in the gene, which may have biological implications with respect to chromatin structure and its function in the cell.

AT Base Pair Anions versus (9-Methyl-A)(1-Methyl-T) Base Pair Anions

The anionic base pairs of adenine and thymine, (AT)(-), and 9-methyladenine and 1-methylthymine, (MAMT)(-), have been investigated both theoretically and experimentally in a complementary, synergistic study. Calculations on (AT)(-) found that it had undergone a barrier-free proton transfer (BFPT) similar to that seen in other dimer anion systems and that its structural configuration was neither Watson-Crick (WC) nor Hoogsteen (HS). The vertical detachment energy (VDE) of (AT)(-) was determined by anion photoelectron spectroscopy and found to be in agreement with the VDE value predicted by theory for the BFPT mechanism. An AT pair in DNA is structurally immobilized into the WC configuration, in part, by being bonded to the sugars of the double helix. This circumstance was mimicked by methylating the sites on both A and T where these sugars would have been tied, viz., 9-methyladenine and 1-methylthymine. Calculations found no BFPT in (MAMT)(-) and a resulting (MAMT)(-) configuration that was either HS or WC, with the configurations differing in stability by ca. 2 kcal/mol. The photoelectron spectrum of (MAMT)(-) occurred at a completely different electron binding energy than had (AT)(-). Moreover, the VDE value of (MAMT)(-) was in agreement with that predicted by theory. The configuration of (MAMT)(-) and its lack of electron-induced proton transfer are inter-related. While there may be other pathways for electron-induced DNA alterations, BFPT in the WC/HS configurations of (AT)(-) is not feasible.

Structures of complexes between echinomycin and duplex DNA

The structure of the bis-intercalation complex of the depsipeptide antibiotic echinomycin with (CGTACG)2 has been redetermined at a higher resolution (1.4 A) and new high-resolution structures (1.1-1.5 A) are reported for the complexes of echinomycin with (GCGTACGC)2 (at both low and high ionic strengths) and (ACGTACGT)2. The structures show the expected Hoogsteen pairing for the base pairs flanking the intercalating chromophores on the outside and Watson-Crick pairing for both base pairs enclosed by the echinomycin. In the octamer complexes but not the hexamer complex, the echinomycin molecule, which would possess a molecular twofold axis were it not for the thioacetal bridge, shows twofold disorder. In all the structures the stacking of the base pairs and chromophores is extended by intermolecular stacking. The structures provide more precise details of the hydrogen bonding and other interactions between the bis-intercalating antibiotics and the duplex DNA than were previously available.

Ability of thrombin to act as molecular chaperone, inducing formation of quadruplex structure of thrombin-binding aptamer

Single-stranded guanosine (G)-rich oligonucleotides have a propensity to form quadruplex structures stabilized by 4-G quartets. The four guanines of such a quartet array in a square planar conWguration, each G interacting with the adjacent one via two hydrogen bonds and behaving as H acceptor/donor of what is known as a Hoogsteen basepair. These structures are believed to exist only in the presence of certain cations, described to stabilize the quadruplex, presumably by compensating the intermolecular repulsion due to the negative charges of the nucleic acid [1–3]. Proteins able to bind such structures under physiological conditions have been described [3,4], and two of them have even been suggested to promote quadruplex formation [5,6]. The fact that some quadruplex-forming sequences have been described to inhibit proliferation of viruses or cancer cells is being exploited for its potential therapeutic application [1]. The thrombin-binding aptamer (TBA)1 was the Wrst in vitro-selected oligonucleotide targeted toward a protein that has no known physiological binding to nucleic acids. The TBA is known to be in equilibrium between a random and a quadruplex conformation when dissolved in aqueous solution [7]. This quadruplex structure contains two G(syn)-G(anti)-G(syn)-G(anti) quartets and three lateral loops, in what is usually referred to as a “chair structure” [8–11]. The presence of K+ in the medium was believed to be necessary to shift the equilibrium toward the quadruplex conformation, subsequently

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 η

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.