A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides

Abstract

Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate. The Klenow fragment of E. coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold. Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2'-OH of an incoming rNTP. A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 "steric gate" can be fully effective. Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides.

Figure 1

A stereo view of the Klenow fragment of DNA polymerase I (Kf pol) structure (4). The separate 3′-5′ exonuclease domain is indicated, as are the fingers and thumb subdomains that form the sides of the polymerase cleft. Within the polymerase domain are shown two long helices: O, on the fingers subdomain, and Q, connecting fingers and palm subdomains. The side chains shown in green indicate the positions of mutations that had little or no effect on dNTP/rNTP discrimination (Table 1); those shown in red correspond to mutations whose phenotypes are discussed in the text. Side chains shown in purple are the important catalytic residues, Asp-705, Asp-882, and Lys-758, which could not be screened because the relevant mutant proteins had too little activity. This figure was made by Jimin Wang, using molscript (5).

Figure 2

Gel assay of dNTP/rNTP discrimination. (A) Comparison of wild-type and E710A Kf pol at various rNTP:dNTP ratios, indicated above each set of four lanes. A ratio of 1000:1 corresponded to 1.5 mM rNTP (U, C, G, or A, as indicated) and 1.5 μM of each dNTP in the reaction. For 10:1 and 1:1 ratios, dNTPs were present at 13 μM. P indicates the position of the labeled primer. Comparison of the products shown here with samples taken before alkali digestion confirmed that the bands seen represent bona fide ribonucleotide incorporation. (B) Comparison of various mutant derivatives of Kf pol under the same conditions as in A, except that the rNTP:dNTP ratio was 200:1, with each of the four dNTPs at 1.5 μM. The data in B were taken from several gels; hence, corresponding bands are not necessarily in identical positions.

Figure 3

Dependence of reaction rate on Mg²⁺ concentration for wild-type and E710A Kf pol. The single-turnover rate for rCMP addition to the C-substrate (Fig. 4A) was measured at a series of Mg²⁺ concentrations from 0.25 to 50 mM, with rCTP at 0.2 μM (▴, broken lines). (The Mg²⁺ optimum was essentially the same when the experiment was repeated at a different rCTP concentration.) The Mg²⁺ dependence for dTMP incorporation by these two proteins (•, solid lines) is taken from steady-state measurements; as we have argued previously (11), the comparison of pre-steady-state and steady-state data appears to be justified in this case. For each plot, the maximal rate is arbitrarily set at 1.

Figure 4

Addition of successive rNTPs. (A) The two DNA substrates used in this experiment and in the kinetic studies. (B) Extension of the 20-mer primers (P) by wild-type and E710A Kf pol in the presence of all four rNTPs at 83 μM. Samples were taken at the indicated times (hr); the 15-hr sample was supplemented with fresh rNTPs (100 μM) and polymerase after 2 hr of incubation. C denotes a portion of the 2-hr sample that was chased with dNTPs (60 μM) and wild-type Kf pol (≈3 μM) for 13 hr; an aliquot of this reaction mixture was then digested with piperidine (D).

Figure 5

Gel assay of dNTP/3′-dNTP discrimination by wild-type and various mutant derivatives of Kf pol. A single 3′-dNTP (U, C, or A, as indicated), which acts as a chain terminator, competes with the corresponding dNTP during elongation of the primer (P). The ratio of 3′-dNTP to dNTP was 20:1.