Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[a]pyrene dG adduct

Abstract

DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N(2)-deoxyguanine adducts efficiently and accurately. The unique features of Polκ, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polκ variants, which have reduced gap size on both sides [Polκ Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polκ variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N(2)-dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polκ-deficient cells stably complemented with PGM1 GFP-Polκ remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polκ restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polκ (mPolκ(52-516)) leads to reduced polymerization activity, and the mutant PGM2(52-516) but not PGM1(52-516) can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ(52-516) retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polκ during translesion synthesis.

Keywords: polycyclic aromatic hydrocarbons; translesion DNA synthesis.

Fig. 1.

Design of PGM1 and PGM2 mutant mPolκ. (A) A back view of the structure of hPolκ–DNA–dNTP complex structure (PDB ID code 2OH2). Polκ is shown in gold ribbon diagram and covered with a semitransparent molecular surface. The residues to be mutated are shown in ball-and-stick model. The DNA template strand is shown in magenta and the template base is highlighted in red opposite the incoming dNTP (cyan). (B) Structure of Dpo4–DNA–dNTP complex (PDB ID code 2AGQ). After superposition with Polκ, Dpo4 is shown in blue with the corresponding residues along the structural gap highlighted in ball-and-stick models. (C) Models of PGM1 and (D) PGM2 mutant mPolκ. The mutated residues are highlighted in blue and labeled according to mPolκ residue number. The corresponding human residues are exactly one number higher (Fig. S1).

Fig. 2.

Primer extension opposite normal and BPDE-dG templates by full-length WT, PGM1, and PGM2 mouse Polκ. (A) Structures of BP, its (+)-(7R,8S,9S,10R) dihydrodiol epoxide metabolite [(+)-anti-BPDE], and the BPDE-dG [10S (+)-trans-anti-[BP]-N²-dG (2)] adduct in DNA. (B) Reactions were carried out in the presence of all four dNTPs for 5 min at 37 °C with increased concentrations of Polκ as shown below each track. (C) Analysis of extension product lengths over normal dG template by PGM1, PGM2, and WT mPolκ. The 43-mer marker is in a separate lane. The reactions were catalyzed by 10 nM Polκ at 37 °C for 15 min. (D) Running-start primer extension experiments on BPDE-dG template. Reactions were carried out with increasing concentrations of Polκ at 37 °C for 15 min. The underlined “G” represents the BPDE-dG lesion. The 22-mer primer was readily extended to the two nucleotides 3′ of the lesion and stalled at template position −1, immediately before the lesion. (E) Comparison of primer extension products length catalyzed by PGM1, PGM2, and WT mPolκ by using the BPDE-dG template. The assays included 50 nM of each Polκ at 37 °C for 30 min.

Fig. 3.

PGM1 GFP-Polκ has a defect in complementation of BPDE hypersensitivity of Polκ-deficient MEFs. The Polκ-deficient MEFs stably complemented with GFP or WT, PGM1, and PGM2 GFP-Polκ were treated with UV (A) or BPDE (B) as described in SI Materials and Methods. Surviving fractions are expressed as a percentage of mock-treated cells. Values are the mean of three independent experiments (±SE).

Fig. 4.

N-terminal truncated Polκ and their activities in normal and TLS. (A) A front view of the hPolκ–DNA–dNTP complex structure (PDB ID code 2OH2). Residues 32 to 51 of the N-clasp (green) connect the catalytic core (bright orange) with LF (pale yellow), and, in the second part of the N-clasp (52–66 aa), Lys55 and Arg62 directly interact with the primer strand. (B) Deletion of residues 1 to 51 in the N-clasp is predicted to result in loss of hydrophobic interactions of the N-clasp with the finger and LF domains. (C) In PGM2, the requirement of 1 to 51 aa to connect the catalytic core and LF is predicted to be substituted by the long loop inserted (blue) in the finger domain. (D) DNA synthesis activities over normal dG template. Reactions were carried out with 20 nM DNA substrate and increasing concentration of mPolκ_52–516 at 37 °C for 15 min. (E) Primer extension on BPDE-dG damaged template. A total of 10 nM DNA was incubated with different concentrations of PGM2 and WT mPolκ_52–516 at 37 °C for 15 min. The reaction with WT Polκ is described in Fig. 2D.

Fig. 5.

A molecular model of BPDE accommodation and bypass by Polκ. (A) Atomic structure of the (+)-trans-anti-BPDE-dG determined by NMR (40, 41). (B) The BPDE adduct is situated in the minor groove and points toward the 5′ end at a template and primer junction (PDB ID code 1AXO). (C) In the apo-hPolκ_68–517 structure (PDB ID code 1T94), the LF is flexible and far removed from the catalytic core. (D) Both DNA and Polκ need to undergo conformational changes as indicated by arrows in B and C, to accommodate the BPDE-dG lesion in the minor groove for TLS. The adduct is predicted to point toward the 3′ end of the template strand and form van der Waals contacts with Phe170. (E) In the PGM1 complex, the aromatic rings of BPDE would clash with F170W and the sugar moiety of BPDE would clash with S131R. (F) In the PGM2 complex, the sugar moiety of BPDE would clash with the loop transplanted from Dpo4 (shown in blue). The rest of the large structural gap likely remains intact.