Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η

Abstract

Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10(3)-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.

Keywords: DNA damage; DNA enzyme; DNA polymerase; enzyme kinetics; translesion synthesis; x-ray crystallography.

FIGURE 1.

hpol η incorporates dNTPs or rNTPs opposite an unmodified DNA template. A, full-length extension of the primer opposite unmodified DNA template (5 μm) with all four dNTPs or rNTPs by hpol η (1.2 μm) at 37 °C for 5, 30, 55, and 240 min (time gradients depicted with wedges). B, single nucleotide incorporation assays with 5 μm native primer-template DNA substrate, 500 nm hpol η, and 1 mm each of individual dNTP or rNTP at 37 °C for 5, 30, and 55 min.

FIGURE 2.

hpol η can bypass an 8-oxodG lesion and incorporate dNTPs or rNTPs. A, hpol η (1.2 μm) extended the primer against DNA template (5 μm) containing an 8-oxoG lesion in the presence of all four dNTPs or rNTPs at 37 °C for 5, 30, 55, and 240 min (time gradients depicted with wedges). B, single nucleotide incorporation assays with hpol η (500 nm) with 5 μm DNA substrate with 8-oxoG in the template and 1 mm each of dNTP or rNTP at 37 °C for 5, 30, and 55 min.

FIGURE 3.

hpol η can incorporate ribonucleotides opposite the CPD lesion and further extend the primer. A, extension of the primer opposite a DNA template (5 μm) containing a CPD lesion by hpol η (1.2 μm) in the presence of all four dNTPs or rNTPs at 37 °C for 5, 30, 55, and 240 min (time gradients depicted with wedges). B, extension of the primer by incubation of 5 μm DNA substrate with a CPD in the template strand, 500 nm hpol η, and 1 mm each of individual dNTP or rNTP at 37 °C for 5, 30, and 55 min.

FIGURE 4.

Quality of the final Fourier (2F_o − F_c) sum electron density drawn at the 1σ threshold around incoming ribonucleotides and the template base/lesions. A, hpol η·dG:rCTP. B, hpol η·(8-oxodG):rCTP. C, hpol η·dG:rATP.

FIGURE 5.

Crystal structure of hpol η inserting rCTP opposite template dG in the presence of Ca²⁺. A, active site of the hpol η·dG:rCTP complex viewed from the major groove side. B, superimposition of the structures of the ternary hpol η·dG·rCTP and hpol η·dG·dCMPNPP (dCTP analog) (PDB code 4O3N) (29) complexes, viewed from the major groove. C, dG:rCTP pair at the active site viewed from the top. D, top view of the superimposed active sites in the structures of hpol η·dG:rCTP and hpol η·dG:dCMPNPP complexes. For hpol η·dG:rCTP, the base pair dG:rCTP (as well as Ca²⁺) are highlighted in dark cyan, and the other nucleotides and key residues Arg-61, Gln-38, and Phe-18 are in light green. For hpol η·dG:dCTP, both the base pair G:dCMPNPP and Mg²⁺ are shown in orange, and the other nucleotides and Phe-18 are shown in khaki.

FIGURE 6.

Crystal structure of hpol η inserting rCTP opposite 8-oxodG. A, active site of the hpol η·(8-oxodG anti):rCTP complex viewed from the major groove side. B, superimposition of the structures of the ternary hpol η-(8-oxodG anti):rCTP and hpol η·(8-oxodG):dCMPNPP (PDB code 4O3P) (29) complexes, viewed from the major groove. C, (8-oxodG anti):rCTP pair at the active site viewed from the top. D, top view of the superimposed active sites in the structures of the hpol η·(8-oxodG anti):rCTP and hpol η·(8-oxodG):dCMPNPP complexes. E, active site of the hpol η·(8-oxodG syn):rCTP complex viewed from the major groove side. F, superimposition of the structures of the ternary hpol η·(8-oxodG syn):rCTP and hpol η·(8-oxodG):dCMPNPP (PDB code 4O3P)(29) complexes, viewed from the major groove. G, (8-oxodG syn):rCTP pair at the active site viewed from the top. H, top view of the superimposed active sites in the same structures of the hpol η·(8-oxodG syn):rCTP and hpol η·(8-oxodG):dCMPNPP complexes. The color codes are the same as in Fig. 5 and H-bonds are dashed lines.

FIGURE 7.

Crystal structure of hpol η inserting rATP opposite 8-oxodG. A, active site of the hpol η·(8-oxodG):rATP complex viewed from the major groove side. B, superimposition of the structures of the ternary hpol η·(8-oxodG):rATP and hpol η·(8-oxodG):dAMPNPP (PDB code 4O3O) (29) complexes. C, (8-oxodG):rATP pair at the active site viewed from the top. D, top view of the superimposed active sites in the structures of the hpol η·(8-oxodG):rATP and hpol η·(8-oxodG):dAMPNPP complexes. The color codes are the same as in Fig. 5.

FIGURE 8.

Second line of defense in hpolη: Tyr-92 stabilizes the steric gate residue Phe-18 by π-π interaction. A, steric gate residue Phe-18 and the second line of defense residue Tyr-92 in the hpol η·dG:rCTP complex viewed from the side. B, view of Phe-18 and Tyr-92 in the superimposition of the structures of hpol η·dG:rCTP and hpol η·dG:dCMPNPP (PDB code 4O3N) (29). In hpol η·dG:rCTP, Phe-18 and Tyr-92 are shown in green, and rCTP and Ca²⁺ are in dark cyan. In hpol η·dG:dCMPNPP, Phe-18 and Tyr-92 are shown in khaki, and dCMPNPP and Mg²⁺ in shown in orange.