Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro

Abstract

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

Figure 1

Analyses of purified human Polκ. (A) Purified human Polκ (200 ng) was analyzed by electrophoresis on a 10% SDS–polyacrylamide gel and visualized by silver staining. Protein size markers (lane M) are indicated on the left. (B) Purified human Polκ (40 ng) was analyzed by a western blot using a mouse monoclonal antibody against the His₆ tag. Protein size markers (lane M) are indicated on the right. (C) DNA polymerase assays were performed without (lane 1) or with (lane 2) purified human Polκ (0.2 ng, 2 fmol), using the 30mer DNA template, 5′-CCTTCTTCATTGGAACATACTTCTTCTTCC-3′, annealed with the 5′-³²P-labeled primer, 5′-GGAAGAAGAAGTATGTT-3′. DNA size markers in nucleotides are indicated on the right.

Figure 2

Bypass of a template 8-oxoguanine (8-oxoG) by human Polκ. Standard DNA polymerase assays were performed with purified human Polκ (0.4 ng, 4 fmol) using the indicated DNA substrate containing a template 8-oxoG and a ³²P-labeled 17mer primer. Polymerase reactions were carried out in the presence of a single deoxyribonucleoside triphosphate dATP (lane 2), dCTP (lane 3), dTTP (lane 4) or dGTP (lane 5), or all four dNTPs (lane 1). DNA size markers in nucleotides are indicated on the left.

Figure 3

Bypass of a template AP site by human Polκ. (A) DNA templates used for AP site bypass assays. The 17mer primer was labeled with ³²P at its 5′-end as indicated by an asterisk. The AP site is located at the X position. Different template bases 5′ to the AP site are underlined. The AflII restriction cleavage site on the ³²P-labeled strand is shown, as well as the AflII recognition sequence. (B) Standard polymerase assays were performed with 20 ng (200 fmol) of purified human Polκ using an undamaged DNA template (lane 1) or the various AP site-containing templates as indicated (lanes 2–5). (C) Polymerase assays were performed using undamaged template 18T (lane 1) or the AP site-containing template AP–T (lane 2) containing the ³²P-labeled primer, 5′-CGCGCGAAGACCGG-3′. This ‘running start’ primer was annealed 3 nt before the AP site (arrowhead) on template AP–T. Polκ used was 10 ng (100 fmol) and 20 ng (200 fmol) for lanes 1 and 2, respectively. DNA size markers in nucleotides are indicated on the left.

Figure 4

Nucleotide incorporation opposite the template AP site. Lesion bypass assays were performed with various AP site-containing templates (50 fmol) in a standard DNA polymerase reaction buffer containing all four dNTPs (lane 1) or dATP (lane 2), dCTP (lane 3), dTTP (lane 4) and dGTP (lane 5) individually. Templates contained a ³²P-labeled 17mer primer annealed right before the template AP site. DNA sequences of the various templates are shown in Figure 3A. (A) Template AP–T, 2 ng (20 fmol) Polκ; (B) template AP–G, 20 ng (200 fmol) Polκ; (C) template AP–A, 20 ng (200 fmol) Polκ; (D) template AP–C, 20 ng (200 fmol) Polκ. DNA size markers in nucleotides are indicated on the left. Quantitation of extended primers is shown below the gels.

Figure 5

Analysis of AP site bypass products by AflII restriction digestion. (A) Standard DNA polymerase assays were performed with the undamaged DNA template (Temp 18T) or the AP site-containing template (AT–T) using 20 ng (200 fmol) of human Polκ. After the polymerase reaction, 5 µl of the reaction products were mixed with 2 µl of H₂O, 1 µl of the 10× AfIII buffer (500 mM Tris–HCl pH 8.0, 100 mM MgCl₂) and 2 µl of AfIII (20 U). AfIII digestions were at 37°C for 4 h. The digested products were separated by electrophoresis on a 20% denaturing polyacrylamide gel and visualized by autoradiography. Samples without (AflII, –) or with (AflII, +) AflII treatment are indicated. DNA size markers in nucleotides are indicated on the left. (B) Standard DNA polymerase assays were performed with the AP site-containing templates as indicated using 20 ng (200 fmol) of human Polκ. After the polymerase reaction, 5 µl of the reaction products were digested with AflII as in (A). DNA sequences of the various templates are shown in Figure 3A.

Figure 6

Bypass of AAF-guanine by human Polκ. (A) DNA polymerase assays were performed with purified human Polκ using undamaged template (lane 1) or the AAF-damaged template (lane 2), both of which shared identical nucleotide sequence. The ³²P-labeled ‘running start’ primer was annealed 4 nt before the AAF-G as indicated. Polκ used was 10 ng (100 fmol) and 20 ng (200 fmol) for lanes 1 and 2, respectively. The 18mer DNA band extended opposite the template AAF-G is indicated by an arrowhead. (B) DNA polymerase assays were performed with purified human Polκ (10 ng, 100 fmol) using the indicated DNA substrate containing a template AAF-G and a ³²P-labeled 17mer primer. Polymerase reactions were carried out in the presence of a single deoxyribonucleoside triphosphate dATP (lane 3), dCTP (lane 4), dTTP (lane 5) or dGTP (lane 6) or all four dNTPs (lane 2). Lane 1, control reaction without Polκ. DNA size markers in nucleotides are indicated on the left.

Figure 7

Bypass of a template (–)-trans-anti-BPDE-N²-dG lesion by human Polκ. A 19mer primer was labeled with ³²P at its 5′-end and annealed right before a template (–)-trans-anti-BPDE-N²-dG as shown on the right. Polymerase reactions were performed with 2 ng (20 fmol) of human Polκ in the presence of a single deoxyribonucleoside triphosphate dATP (lane 2), dCTP (lane 3), dTTP (lane 4) or dGTP (lane 5) or all four dNTPs (lane 1). DNA size markers in nucleotides are indicated on the left.

Figure 8

Response of human Polκ to a TT dimer and a TT (6-4) photoproduct in template DNA. Standard DNA polymerase assays were performed using the indicated DNA substrates containing a template TT dimer or a TT (6-4) photoproduct and a ³²P-labeled 15mer primer. The modified template TT sequence is indicated. Polymerase reactions were carried out with 0.8 ng (10 fmol) of purified human Polη (lane 2) or 20 ng (200 fmol) of purified human Polκ (lanes 3 and 4). Lane 1, control reaction without DNA polymerase. DNA size markers in nucleotides are indicated on the left.