Minor Groove Interactions between Polymerase and DNA: More Essential to Replication than Watson−Crick Hydrogen Bonds?

Abstract

Studies of the enzymatic mechanisms responsible for DNA synthesis and mutations have often focused on the structural and energetic issues of base pair geometry and Watson–Crick hydrogen (H) bonding.1 Until relatively recently, it was thought that a polymerase was not likely to make specific H-bonded interactions with individual DNA bases since the four base structures differ significantly, although earlier work pointed out that H-bond acceptors appear at similar positions for A–T and G–C in the minor groove.2 Recent X-ray crystal structures of DNA polymerases bound to duplexes,3 however, have implicated amino acid side chains in H bonding to minor groove acceptor atoms, and thus as potentially important to insertion and extension of base pairs during DNA replication. For example, in the Bacillus stearothermophilus DNA polymerase I large fragment,3c which is highly homologous to the Klenow fragment of DNA polymerase I from Escherichia coli, conserved protein side chains or oriented water molecules anchored to them, form H bonds to the first four base pairs extending from the 3′-primer terminus at N3 of purines and O2 of pyrimidines. Mutagenesis studies of the Klenow fragment (Kf) of E. coli DNA polymerase I, human DNA polymerase β (Pol β) and HIV-1 reverse transcriptase have examined the importance to replication of some of the analogous residues.4 Alanine substitutions of Arg 668 and Gln 849 in Kf and alanine and leucine substitutions of Asn 279 and Arg 238 in Pol β, all thought to be minor groove H-bond donors, markedly decrease DNA-binding affinity and the kcat for DNA synthesis,4a,b,c although which groups on the DNA are affected is not clear. To test these effects, we synthesized a new 9-methyl-1-H-imidazo[(4,5)-b]pyridine nucleoside analogue, 1 (denoted Q), which is isoelectronic and isosteric with deoxyadenosine (Figure 1A). It lacks all Watson–Crick H bonding groups, but does have a minor groove acceptor nitrogen, analogous to N3 of adenine. For comparison, we also made use of a 4-methylbenzimidazole deoxynucleoside 2 (denoted Z),5 which differs from Q only by the absence of N3; it lacks both Watson–Crick pairing and minor groove acceptor ability. For geometric complementarity, we used nucleoside F (Figure 1A), which is a thymidine isostere,6,7 as a pairing partner, and pairs with natural bases were examined as well. We evaluated the effects with the Klenow polymerase (lacking 3′-5′ exonuclease activity), which is among the best characterized DNA polymerases. Although previous studies have evaluated the importance of Watson–Crick H bonding to nucleotide insertion, the present studies are the first to evaluate minor groove effects for both sides of the minor groove using the same analogues, and both for insertion and extension of base pairs. Figure 1 Structures and sequences in this study. (A) Structures of natural DNA bases and analogues in this study. (B) Sequences of primer and template DNAs used in the polymerase experiments. Single-nucleotide insertions were carried out to qualitatively evaluate minor groove effects on the ability of the polymerase to form a base pair. A 5′-32P-labeled 23mer (Figure 1B) was extended one base by inserting nucleotides opposite A, Z, and Q nucleosides in the template by the polymerase. Results evaluated by gel electrophoresis show that dFTP is efficiently inserted opposite Z and Q (Figure 2A, lanes 10 and 15) and that there is a small difference between the two. Among the natural nucleoside triphosphates, dTTP shows some insertion opposite Z and also opposite Q, although to a lesser extent. The other nucleotides are very poorly inserted opposite these nonpairing bases. Overall, the data suggest that the presence or absence of a minor groove acceptor atom in the template strand makes only a small difference in the ability to insert a nucleotide opposite that base. Figure 2 Autoradiograms of denaturing PAGE gels showing minor groove effects on nucleotide insertion. (A) Minor groove interaction effects on single-nucleotide insertions opposite A, Z, and Q in the template. (B) Minor groove interaction effects on single-nucleotide ... To test minor groove effects for the incoming nucleotide itself, we also synthesized the nucleoside triphosphate analogue of Q (dQTP) and compared it to dZTP and dATP in its ability to be inserted opposite each of the four natural bases as well as opposite F, Z, and Q in separate template–primer duplexes (Figure 2B). The results showed selective and relatively efficient nucleotide insertion for dZTP or dQTP opposite F in the template, and almost no insertion opposite the natural bases. The results suggest, therefore, that minor groove interactions have a qualitatively small effect overall for the base pair being formed, in contrast to previous findings (see below). The nonpolar Q–F pair behaves similarly to a Z–F pair, despite the latter having little possibility of H bonding between the polymerase and th