An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a 'steric gate' residue for discrimination against ribonucleotides

Abstract

DNA polymerases of the A and B families, and reverse transcriptases, share a common mechanism for preventing incorporation of ribonucleotides: a highly conserved active site residue obstructing the position that would be occupied by a 2' hydroxyl group on the incoming nucleotide. In the family Y (lesion bypass) polymerases, the enzyme active site is more open, with fewer contacts to the DNA and nucleotide substrates. Nevertheless, ribonucleotide discrimination by the DinB homolog (Dbh) DNA polymerase of Sulfolobus solfataricus is as stringent as in other polymerases. A highly conserved aromatic residue (Phe12 in Dbh) occupies a position analogous to the residues responsible for excluding ribonucleotides in other DNA polymerases. The F12A mutant of Dbh incorporates ribonucleoside triphosphates almost as efficiently as deoxyribonucleoside triphosphates, and, unlike analogous mutants in other polymerase families, shows no barrier to adding multiple ribonucleotides, suggesting that Dbh can readily accommodate a DNA-RNA duplex product. Like other members of the DinB group of bypass polymerases, Dbh makes single-base deletion errors at high frequency in particular sequence contexts. When making a deletion error, ribonucleotide discrimination by wild-type and F12A Dbh is the same as in normal DNA synthesis, indicating that the geometry of nucleotide binding is similar in both circumstances.

Figure 1

Kinetics of dGTP incorporation by F12A Dbh. The first-order rate constants (k_obsd) for dGTP addition to the DNA duplex A (Table 1) were plotted against dGTP concentration. The solid line represents the best fit of the data to a hyperbolic equation, giving a dissociation constant (K_d) of 0.87 ± 0.11 mM for binding of dGTP to the complex of F12A Dbh with primer/template, and a maximum incorporation rate (k_pol) of (1.7 ± 0.1) × 10^–3 s^–1.

Figure 2

Incorporation of dGTP and rGTP by wild-type and F12A Dbh at 50°C, using DNA duplex A (Table 1). Reactions were carried out with 3 mM dGTP or rGTP using the conditions described in Materials and Methods. (A) Polyacrylamide-urea denaturing gel of samples removed and quenched at the indicated times (min). ‘p’ marks the position of the unextended primer. In the addition of dGTP by wild-type Dbh (WT), some primers have incorporated multiple G residues, either by misinsertion or slippage. (B) Quantitation of the gel shown in (A). The extent of reaction is measured as the fraction of primers that have been extended by at least one nucleotide.

Figure 3

Addition of successive rNTPs or dNTPs to DNA duplex A (Table 1) by wild-type (WT) and F12A Dbh and E710A Klenow fragment (KF). Reactions were carried out as described in Materials and Methods, using all four dNTPs or rNTPs at 100 µM (F12A Dbh and E710A KF) or 500 µM (WT Dbh). Samples were removed and quenched at the indicated times (min), and fractionated on a polyacrylamide-urea denaturing gel. ‘p’ marks the position of unextended primer and an asterisk indicates the position of full-length product, corresponding to the addition of 10 nt. In the F12A reactions, faint bands larger than the expected fully extended primer were visible, presumably due to slippage or non-templated addition. These bands were more noticeable in the reaction of WT Dbh, due to the overall faster reaction rate.

Figure 4

Comparison of the structural determinants of sugar selectivity in four DNA polymerase families. Amino acid side chains close to the incoming dNTP in polymerase–DNA–dNTP ternary complexes are illustrated. In each case the steric gate residue that blocks the 2′-OH of an incoming rNTP is shown in purple, and the closest approach of this side chain to the C2′ position is indicated in black (Å). Other side chains (most of which are highly conserved in their respective polymerase families) that appear to play a role in maintaining the geometry of the dNTP binding pocket are shown in cyan. Important hydrogen bonds are illustrated in green. The polymerases are: (A) Dpo4, PDB file 1JX4 (4); (B) Klentaq, PDB file 3KTQ (14); (C) HIV-1 reverse transcriptase, PDB file 1RTD (16); (D) RB69 DNA polymerase, PDB file 1QSS (15). Note that in the Dpo4 structure (A) the nucleotide shown is a nucleoside diphosphate instead of a nucleoside triphosphate, due to hydrolysis during crystallization; moreover, the interaction between the α-phosphate and the polypeptide backbone resembles the interaction involving the β-phosphate in the other three illustrated structures. This figure was made using Swiss PDB viewer (version 3.7b2) (38).

Figure 5

Comparison of the positions of the incoming nucleotide and the Tyr12 steric gate side chain in the Dpo4.1 and Dpo4.2 complexes. The Dpo4.1 complex, in which the incoming nucleotide (ddADP) is paired opposite the first unpaired template base, is illustrated in gray. The Dpo4.2 complex, in which an incoming ddGTP is paired opposite the base 5′ to the first unpaired template base (an apparently frameshifted conformation) is shown in red. Also shown are the primer terminus and the preceding nucleotide from the Dpo4.1 complex, and the primer terminus from the Dpo4.2 complex. The Dpo4.2 complex contains three metal ions at the polymerase active site: two Mg²⁺ (pale green) and one Ca²⁺ (dark green). The Dpo4.1 complex contains only the Ca²⁺ ion in essentially the same position as illustrated for Dpo4.2. The Dpo4.2 complex also contains a molecule of ethylene glycol (purple) that occupies the large space between the primer terminus and the incoming nucleotide. This figure was made by superimposing the alpha carbons from the PDB structure files 1JX4 (Dpo4.1) and 1JXL (Dpo4.2) (4) using Swiss PDB viewer (version 3.7b2) (38).