Role of human DNA polymerase κ as an extender in translesion synthesis

Proteins and DNA Substrates.

Human Polκ was expressed in and purified from yeast strain BJ5464 as described (14). Human Polδ, which was a gift from J. Hurwitz (Memorial Sloan–Kettering Cancer Center, New York), was purified as described (19). Oligonucleotides were synthesized by Midland Certified Reagents (Midland, TX). DNA substrates S1- (shown in Fig. 1) were generated by annealing a 75-nt oligomer template, 5′-AGC TAC CAT GCC TGC CTC AAG AAT TCG TAA XAT GCC TAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-3′, containing a G (S1-G), an m6G (S1-m6G), or a 7,8-dihydro-8-oxoG (S1–8-oxoG) at position 31 (X) to the 5′ ³²P-labeled oligonucleotide primer N4264 (5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA G-3′). For DNA substrate S2- (shown in Fig. 2A and also used for steady-state kinetic analysis of insertion reactions), the 75-nt templates used for generating the substrates S1- were annealed to the 5′ ³²P-labeled oligonucleotide primer N4309 (5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC AT-3′), yielding substrates S2-G, S2–8-oxoG, and S2-m6G. DNA substrates S3- (shown in Fig. 2B and used for steady-state kinetic analysis of extension reactions in Fig. 3B) were generated by annealing the 75-nt oligomer templates used for generating the substrates S1- to the 5′ ³²P-labeled oligonucleotide primers, 5′-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGCATN-3′, where N is G, A, T, or C.

DNA-Polymerase Assays.

A standard DNA-polymerase reaction (10 μl) contained 40 mM Tris⋅HCl (pH 7.5), 8 mM MgCl₂, 1 mM DTT, 100 μg/ml BSA, 10% glycerol, 20 nM 5′ ³²P-labeled oligonucleotide primer annealed to an oligonucleotide template, and 5 or 100 μM of each of all four deoxynucleotides (dGTP, dATP, dTTP, and dCTP) as indicated in the figure legends. Reactions were started by the addition of Polκ (3 nM) or Polδ (0.03 unit, unit is as defined in ref. 19) and incubated at 37°C for 10 min followed by quenching by the addition of loading buffer (40 μl) containing 20 mM EDTA, 95% formamide, 0.3% bromophenol blue, and 0.3% cyanol blue. The reaction products were resolved on 10% polyacrylamide gels containing 8 M urea. Quantitation of the results was done by using a Molecular Dynamics STORM PhosphorImager and IMAGEQUANT software. The concentration of the product of the bypass reaction was calculated as the ratio of the intensity of bands that lie above the lesion site to the intensity of all bands including the primer.

Analysis of Steady-State Kinetics.

Steady-state kinetic analyses for deoxynucleotide incorporation opposite an 8-oxoG or m6G or primer extension opposite from these lesions were performed as described (20–22). Briefly, Polκ (1 nM) was incubated with 10 nM DNA substrate in the presence of increasing concentrations of a single deoxynucleotide for 10 min. The reaction products were resolved on 15% polyacrylamide gels containing 8 M urea. Gel-band intensities of the substrates and products were quantitated by PhosphorImager, and the observed rate of deoxynucleotide incorporation was plotted as a function of dNTP concentration. The data were fit by nonlinear regression using SIGMAPLOT 5.0 to the Michaelis–Menten equation describing a hyperbola, v = (V_max × [dNTP]/(K_m + [dNTP]). The k_cat and K_m steady-state parameters were obtained from the fit and used to calculate the frequency of deoxynucleotide incorporation (f_inc) and the intrinsic efficiency of mispair extension (f) for each primer/template pair by using the equation f_inc or f = (k_cat/K_m)_mispaired/(k_cat/K_m)_paired.

Replication Through the m6G and 8-oxoG Lesions by the Concerted Action of Human Polδ and Polκ.

We examined the ability of human Polκ to replicate past an m6G and an 8-oxoG lesion in the template DNA, and we also carried out bypass reactions by human Polδ and by the combination of Polδ and Polκ. Running-start experiments were performed by using DNA substrates constructed by annealing a linear 75-nt template DNA containing a single m6G, an 8-oxoG, or, as a control, a G residue 45 nt from the 3′ end to a 5′ ³²P-labeled 40-nt primer (Fig. 1), and DNA-synthesis reactions were carried out at low (5 μM) and high (100 μM) nucleotide concentrations. The m6G lesion is a strong block to DNA synthesis, and even at 100 μM dNTP, Polκ replicated through only ≈14% (Fig. 1, lane 10), and Polδ replicated through only ≈4% of this lesion (Fig. 1, lane 12) compared with synthesis on template containing an undamaged G residue (Fig. 1, lanes 3 and 5). Furthermore, Polκ exhibited a strong stall site right before the m6G residue, indicating a strong inhibition of insertion across from this lesion by this polymerase (Fig. 1, lanes 9 and 10). Although Polδ showed some ability to insert nucleotides opposite the m6G lesion, it was not able to subsequently extend from the resulting primer end (Fig. 1, lane 12). Remarkably, however, the combination of Polκ with Polδ promoted m6G bypass, and together they replicated through the m6G lesion ≈50% as efficiently as through the undamaged G residue (Fig. 1, compare lanes 13 and 14 with lanes 6 and 7, respectively).

Polκ replicated through an 8-oxoG residue with ≈50% the efficiency of replicating through an undamaged G (Fig. 1, compare lanes 16 and 17 with lanes 2 and 3, respectively). Moreover, Polκ did not exhibit a prominent stall site just before the 8-oxoG residue or opposite this lesion, suggesting it is able to insert nucleotides opposite the 8-oxoG lesion efficiently and to extend from the inserted nucleotide. Polδ can also replicate through an 8-oxoG lesion, but it does so 3- to 4-fold less efficiently than it replicates through an undamaged G (Fig. 1, compare lanes 18 and 19 with lanes 4 and 5, respectively.) Importantly, however, the most striking increase in 8-oxoG bypass occurred when Polδ was combined with Polκ, and at 5 μM dNTP, the two polymerases together replicated through the 8-oxoG lesion as effectively as through the undamaged G (Fig. 1, compare lanes 20 and 6). The key observation here is that Polδ and Polκ together are much more effective in m6G and 8-oxoG bypass than either polymerase alone. These observations suggested that because of its proximity to the lesion site, Polδ would contribute to the insertion of nucleotides opposite these lesions, whereas Polκ could carry out the subsequent extension from the ensuing primer end.

Nucleotide Incorporation Opposite m6G and 8-oxoG by Human Polδ.

We have shown previously that yeast Polδ inserts a T or C opposite an m6G (10) and an A opposite an 8-oxoG lesion (9), promoting the mutagenic bypass of these lesions. To examine whether human Polδ incorporates the same nucleotides opposite these lesions as yeast Polδ, DNA substrates containing an undamaged G, an m6G, or an 8-oxoG residue next to the primer–template junction were incubated with human Polδ in the presence of just one dNTP. Human Polδ inserted a T or C opposite the m6G lesion (Fig. 2A, lanes 7 and 8), whereas opposite the 8-oxoG lesion, it predominantly inserted an A (Fig. 2A, lane 10), but some C nucleotide was inserted also (Fig. 2A, lane 12).

Extension of Primer Ends Opposite from the m6G and 8-oxoG Lesions by Human Polκ.

Next we examined the ability of Polκ to extend from the various 3′-primer termini situated across from the m6G or 8-oxoG lesion in the 75-nt template in the presence of 100 μM of each of the four dNTPs (Fig. 2B). Polκ extended from the T and C 3′-primer ends situated opposite the m6G site with ≈70% of the efficiency of extension from the C 3′-primer end situated opposite undamaged G (Fig. 2B, compare lanes 7 and 8 with lane 4). The extension from the A or C 3′-primer end opposite from the 8-oxoG residue was efficient also, occurring with ≈70% the efficiency of extension from the C 3′-primer end situated opposite undamaged G (Fig. 2B, compare lanes 10 and 12 with lane 4). Thus, after the insertion of nucleotides opposite the m6G or 8-oxoG lesion by Polδ, Polκ could subsequently extend from the inserted nucleotides.

Steady-State Kinetic Analyses of Nucleotide Insertion and Extension Reactions Across from the m6G and 8-oxoG Lesions by Human Polκ.

To characterize further the role of Polκ in the bypass of m6G and 8-oxoG lesions, we measured the steady-state kinetic parameters of nucleotide insertion and extension reactions opposite from these lesions. The kinetics of insertion of a single deoxynucleotide opposite an m6G and an 8-oxoG (Fig. 3A) and the kinetics of the addition of the next correct nucleotide to various 3′-primer termini situated across from m6G and 8-oxoG (Fig. 3B) were determined as a function of deoxynucleotide concentration under steady-state conditions. We incubated Polκ with m6G- or 8-oxoG-containing DNA substrates and various concentrations of a single nucleotide. The rate of nucleotide incorporation was plotted as a function of nucleotide concentration. The steady-state apparent k_cat and K_m values for each nucleotide incorporation and for extension of each primer terminus were obtained from the curve fitted to the Michaelis–Menten equation by using nonlinear regression. The frequency of nucleotide incorporation, f_inc, and the relative efficiency of extension, f, were calculated as the ratio of the efficiency (k_cat/K_m) of incorrect nucleotide incorporated or extended from to the efficiency (k_cat/K_m) of correct nucleotide incorporated or extended from, respectively (Tables 1 and 2).

Table 1.

Steady-state kinetic parameters of nucleotide-insertion reactions opposite m6G and 8-oxoG template residues by human Polκ

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Table 2.

Steady-state kinetic parameters of extension reactions opposite from m6G and 8-oxoG catalyzed by human Polκ

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As indicated by the k_cat/K_m values shown in Table 1, Polκ incorporated nucleotides opposite the m6G lesion inefficiently. Compared with the incorporation of a C opposite the undamaged G-template residue, Polκ incorporated nucleotides opposite m6G with frequencies ranging from 1 × 10⁻² to 2.6 × 10⁻⁵. Polκ incorporated a T ≈13-fold more efficiently than C opposite m6G; however, even T incorporation opposite m6G was ≈100-fold less efficient than the C incorporation opposite undamaged G (Table 1). In contrast, Polκ extended opposite from the m6G lesion quite efficiently. As shown in Table 2, compared with the extension from a C opposite an undamaged G in the template, Polκ extended from a T and a C opposite from m6G ≈3- and 10-fold less efficiently, respectively. The extension from the A and G nucleotides opposite m6G was much less efficient.

Polκ is quite efficient at inserting an A opposite the 8-oxoG lesion (Table 1). Compared with the incorporation of a C opposite undamaged G in the template, Polκ incorporated an A opposite 8-oxoG only ≈3-fold less efficiently. Polκ also can insert a C opposite 8-oxoG, but it is ≈40-fold less efficient at it than the insertion of C opposite the undamaged G-template residue. In addition, Polκ is an efficient extender opposite from the 8-oxoG lesion (Table 2). Compared with the extension from a C opposite an undamaged G in the template, Polκ extended from an A, C, or T opposite 8-oxoG only ≈19-, 36-, and 43-fold less efficiently, respectively. Overall, Polκ is quite proficient in inserting an A opposite 8-oxoG as well as in extending from this nucleotide.