A novel mechanism of sugar selection utilized by a human X-family DNA polymerase

Abstract

During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2'-hydroxyl group and the bulky side chain of an active-site residue. In this study, we demonstrated that human DNA polymerase lambda used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2'-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such steric clash was further demonstrated to be dependent on the size and orientation of the substituent covalently attached at the ribonucleotide C2'-position.

Figure 1. Sequence alignment of human X-family DNA polymerases and the active site of Polλ

(A) Sequence alignment of the α-helix M in the thumb domain of each human X-family DNA polymerase. Residue numbers correspond to positions with respect to the N-terminal methionines. The black background indicates which residues are conserved among the family members and the arrow head denotes the potential “steric gate” residue. (B) A close-up view of potential steric interactions with the C2′ position of an incoming ddTTP are depicted in the active site of Polλ (PDB 1XSN). Distances are listed in the table for the Y505 side chain and peptide backbone residues Y505-G508 (purple) near the C2′ position.

Figure 1. Sequence alignment of human X-family DNA polymerases and the active site of Polλ

(A) Sequence alignment of the α-helix M in the thumb domain of each human X-family DNA polymerase. Residue numbers correspond to positions with respect to the N-terminal methionines. The black background indicates which residues are conserved among the family members and the arrow head denotes the potential “steric gate” residue. (B) A close-up view of potential steric interactions with the C2′ position of an incoming ddTTP are depicted in the active site of Polλ (PDB 1XSN). Distances are listed in the table for the Y505 side chain and peptide backbone residues Y505-G508 (purple) near the C2′ position.

Figure 2. Concentration dependence on the pre-steady state rate constant of matched ribonucleotide incorporation

(A) Each D-DNA substrate is composed of a 5′-radiolabeled 21-mer, a 5′-phosphorylated 19-mer, and a 41-mer template where ‘X’ represents A for D-1, C for D-8, G for D-6 and D-6T, and T for D-7. The underlined C:G base pair is A:T for D-6T. (B) A pre-incubated solution of Polλ (120 nM) and 5′-[³²P]-labeled D-1 (30 nM) was rapidly mixed with increasing concentrations of UTP·Mg²⁺ (5 μM, ●; 10 μM, ○; 25 μM, ■; 50 μM, □; 100 μM, ▲; 200 μM, △; 400 μM, ◆ and 800 μM, ◇) for various time intervals. The solid lines are the best fits to a single-exponential equation which determined the observed rate constants, k_obs. (C) The k_obs values were plotted as a function of UTP concentration. The data (●) were then fit to a hyperbolic equation, yielding a k_p of 0.053 ± 0.001 s⁻¹ and a K_d of 114 ± 8 μM.

Figure 2. Concentration dependence on the pre-steady state rate constant of matched ribonucleotide incorporation

(A) Each D-DNA substrate is composed of a 5′-radiolabeled 21-mer, a 5′-phosphorylated 19-mer, and a 41-mer template where ‘X’ represents A for D-1, C for D-8, G for D-6 and D-6T, and T for D-7. The underlined C:G base pair is A:T for D-6T. (B) A pre-incubated solution of Polλ (120 nM) and 5′-[³²P]-labeled D-1 (30 nM) was rapidly mixed with increasing concentrations of UTP·Mg²⁺ (5 μM, ●; 10 μM, ○; 25 μM, ■; 50 μM, □; 100 μM, ▲; 200 μM, △; 400 μM, ◆ and 800 μM, ◇) for various time intervals. The solid lines are the best fits to a single-exponential equation which determined the observed rate constants, k_obs. (C) The k_obs values were plotted as a function of UTP concentration. The data (●) were then fit to a hyperbolic equation, yielding a k_p of 0.053 ± 0.001 s⁻¹ and a K_d of 114 ± 8 μM.

Figure 2. Concentration dependence on the pre-steady state rate constant of matched ribonucleotide incorporation

(A) Each D-DNA substrate is composed of a 5′-radiolabeled 21-mer, a 5′-phosphorylated 19-mer, and a 41-mer template where ‘X’ represents A for D-1, C for D-8, G for D-6 and D-6T, and T for D-7. The underlined C:G base pair is A:T for D-6T. (B) A pre-incubated solution of Polλ (120 nM) and 5′-[³²P]-labeled D-1 (30 nM) was rapidly mixed with increasing concentrations of UTP·Mg²⁺ (5 μM, ●; 10 μM, ○; 25 μM, ■; 50 μM, □; 100 μM, ▲; 200 μM, △; 400 μM, ◆ and 800 μM, ◇) for various time intervals. The solid lines are the best fits to a single-exponential equation which determined the observed rate constants, k_obs. (C) The k_obs values were plotted as a function of UTP concentration. The data (●) were then fit to a hyperbolic equation, yielding a k_p of 0.053 ± 0.001 s⁻¹ and a K_d of 114 ± 8 μM.

Figure 3. Model of a ribonucleotide bound at the active site of Polλ

Using the ternary complex Polλ·gapped DNA·ddTTP (PDB 1XSN), the 2′- and 3′-hydroxyl groups were modeled onto the ribose of the ddTTP substrate. The hydroxyl groups were extracted from the ATP substrate in complex with a reverse transcriptase (PDB 2IAJ). The model presented here showed a steric clash between the 2′-hydroxyl of a dTTP analog (rTTP) and the backbone carboxyl group of Y505.