Escherichia coli DNA Polymerase I (Klenow Fragment) Uses a Hydrogen-bonding Fork from Arg668 to the Primer Terminus and Incoming Deoxynucleotide Triphosphate to Catalyze DNA Replication

Abstract

Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg 668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg 668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg 668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1R,3R,4R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter k pol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in k pol . These results are consistent with a hydrogenbonding fork from Arg 668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.The high fidelity of DNA replication synthesis is accomplished despite the similarity in energy between correctly and incorrectly paired bases (1, 2). Since inter-strand hydrogen bonds cannot account for this selectivity (3, 4), other mechanisms have been proposed to supply the fidelity (reviewed in Ref. 5). These include solvation (6), base stacking (7, 8), steric exclusion (3, 4, 9), and minor groove binding (10). These mechanisms are not mutually exclusive and may all enhance selection for the correct base pairs.The minor groove has been suggested to be a site by which polymerases can check geometry because the O 2 -position of pyrimidines and the N-3-position of purines occupy similar spatial orientations as well as being hydrogen bond acceptors (10). Crystal structures of DNA polymerases bound to DNA have shown that there are many interactions between the protein and the minor groove of DNA (11-15). Site-directed mutagenesis studies have implicated several amino acid residues as being important for catalysis and fidelity of DNA replication (15)(16)(17)(18)(19)(20). Similarly, the use of purine analogs such as 3-deazaadenine (21, 22), 3-deazaguanine (3DG) 1 (23, 24), 9-methyl-1H-imidazo[4,5-b]pyridine and 4-methylbenzimidazole (25,26), and the pyrimidine analogs difluorotoluene (25), 2-aminopyridine, and 3-methyl-2-pyridone (27) also have implicated the minor groove of the DNA as being crucial.X-ray crystallographic studies of polymerases that have a structure similar to that of KF Ϫ (Taq (13) Bacillus stearothermophilus (12) and T7 (14)...

Discovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic Alphabet

“…34 The most efficiently extended termini all had a minor groove H-bond acceptor in the primer and a relatively hydrophobic minor groove substituent in the template nucleobase. Biochemical studies of natural DNA have clearly shown that H-bond acceptors in the minor groove of the primer nucleobase facilitate extension; [40][41][42][43] interactions between these functional groups and Kf, particularly H-bonds formed between the minor groove H-bond acceptor of the primer nucleobase, Arg668 of Kf, and the ribosyl oxygen of the incoming dNTP, are necessary for the polymerase to properly orient the primer terminus and the incoming triphosphate for catalysis. 42 We have repeatedly observed the same phenomenon in unnatural scaffolds.…”

“…Biochemical studies of natural DNA have clearly shown that H-bond acceptors in the minor groove of the primer nucleobase facilitate extension; [40][41][42][43] interactions between these functional groups and Kf, particularly H-bonds formed between the minor groove H-bond acceptor of the primer nucleobase, Arg668 of Kf, and the ribosyl oxygen of the incoming dNTP, are necessary for the polymerase to properly orient the primer terminus and the incoming triphosphate for catalysis. 42 We have repeatedly observed the same phenomenon in unnatural scaffolds. [18][19][20][21][22] Here, this idea is further supported by the significantly decreased efficiency of extension of primers terminating in either dMM3 or dDM5.…”

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

“…The sequences of the oligodeoxynucleotides are shown in Table 1. The primer was 32 P-labeled with [␥-32 P]ATP and annealed with a 20% excess of the template as described previously (32). The P15/T24 substrate was used in all except the pyrophosphorylation experiments and is referred to as the DNA substrate.…”

“…This reaction was fit to the burst equation to obtain a reduced amplitude of 3.2 Ϯ 0.3 nM. To determine the amount of product that is caused by the rebinding of [␣- 32 P]dCTP in the presence of 1 mM unlabeled dCTP, the experiment described by Fig. 7A (OE) was performed.…”

“…7A (OE) was performed. In this protocol, pol (60 nM), DNA (300 nM), and dCTP (20 M) were mixed with [␣- 32 P]dCTP (20 M), dCTP (980 M), and 6 mM Mg 2ϩ . As shown in Fig.…”