Unlocking the sugar "steric gate" of DNA polymerases

Abstract

To maintain genomic stability, ribonucleotide incorporation during DNA synthesis is controlled predominantly at the DNA polymerase level. A steric clash between the 2'-hydroxyl of an incoming ribonucleotide and a bulky active site residue, known as the "steric gate", establishes an effective mechanism for most DNA polymerases to selectively insert deoxyribonucleotides. Recent kinetic, structural, and in vivo studies have illuminated novel features about ribonucleotide exclusion and the mechanistic consequences of ribonucleotide misincorporation on downstream events, such as the bypass of a ribonucleotide in a DNA template and the subsequent extension of the DNA lesion bypass product. These important findings are summarized in this review.

Figure 1

Structural basis for ribonucleotide exclusion. The ‘steric gate’ residues (green sticks) and other nearby active site residues (lavendar sticks) are shown with the incoming nucleotide (gray sticks) for (A) Taq (PDB 3KTQ), (B) RB69 (PDB 1IG9), (C) HIV-1 RT (PDB 1RTD), (D) Dpo4 (PDB 2AGQ), (E) human Pol β (PDB 1BPY), and (F) mouse Pol μ (PDB 2IHM). The C2′ position is indicated using an arrow for each nucleotide. The shortest distance between the ‘steric gate’ and C2′ position is indicated with a red dashed line. Hydrogen bonding interactions are shown as a black dashed line while potential hydrogen bonds with the missing 3′-OH are shown as a green dashed line.

Figure 2

Measurements of sugar selectivity for ‘steric gate’ mutants. The sugar selectivity values of WT (dark bars) and mutant (gray bars) DNA polymerases and RTs were obtained from the references listed in Table 1. The mutations are as follows: E710A for KF, Y254V for Phi 29, Y416A for RB69, Y505A for hPol λ, G433Y for hPol μ, F12A for Dbh, Y12A for Dpo4, Y115A for HIV-1 RT, and F155V for MMLV RT. The incoming dNTP and rNTP pair are indicated in parenthesis.

Figure 3

Structural models of a ribonucleotide in the active site of Dpo4. The ‘steric gate’ residue (green sticks) and incoming nucleotide (gray sticks) are shown for (A) WT Dpo4 with dATP (PDB 2AGQ), (B) WT Dpo4 with rATP, and (C) Dpo4 Y12A with rATP. The ribose ring of rATP in (B and C) is from PDB 3ETH while the side chain of Y12 is from PDB 2AGQ and was replaced with Ala in (C) using SwissPDB Viewer (36). The 2′ position is indicated by an arrow.

Figure 4

Mutations mapped onto the crystal structure of a truncated form of Taq (PDB 3KTQ). Residues that were mutated (blue space-filling models) are labeled on the ternary complex of Taq (gray), DNA (black), and ddNTP (green sticks). The ‘steric gate’ residue E615 is in pink and the C2′ position is identified by an arrow.