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. 2000 Jul 1;14(13):1589-94.

Error-prone Bypass of Certain DNA Lesions by the Human DNA Polymerase Kappa

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Free PMC article

Error-prone Bypass of Certain DNA Lesions by the Human DNA Polymerase Kappa

E Ohashi et al. Genes Dev. .
Free PMC article

Abstract

The Escherichia coli protein DinB is a newly identified error-prone DNA polymerase. Recently, a human homolog of DinB was identified and named DINB1. We report that the DINB1 gene encodes a DNA polymerase (designated polkappa), which incorporates mismatched bases on a nondamaged template with a high frequency. Moreover, polkappa bypasses an abasic site and N-2-acetylaminofluorene (AAF)-adduct in an error-prone manner but does not bypass a cis-syn or (6-4) thymine-thymine dimer or a cisplatin-adduct. Therefore, our results implicate an important role for polkappa in the mutagenic bypass of certain types of DNA lesions.

Figures

Figure 1
Figure 1
Detection of DNA polymerase activity. (A) A schematic representation of the E. coli DinB, human DINB1, and XPV proteins. Motifs I–V are shared among the UmuC/DinB family proteins, but motif VI is found only in the DinB subgroup and motif Ia is conserved among mammalian and C. elegans DinB homologs (Ogi et al. 1999). Motifs VIIa and VIIb denote Zinc clusters (Gerlach et al. 1999). (B) SDS–PAGE analysis of purified proteins. The purified proteins (350 ng each) were analyzed by electrophoresis on a 10% SDS-PAGE with marker proteins (New England Biolabs) and visualized by CBB staining. (C) DNA polymerase activity. Four different amounts of DINB1ΔC and DINB1ΔC -D198N (0.04, 0.4, 4, and 40 nm from left to right) were added in the reaction mixture. After incubation at 37°C for 15 min, the reaction products were analyzed by electrophoresis on 20% polyacrylamide/7m urea sequencing gel followed by autoradiography.
Figure 2
Figure 2
Fidelity of polκΔC on nondamaged DNA template. The 30mer with the indicated sequence (A–D) or with the G-to-T substitution (E) was annealed with a 5′-labeled primer of different length. The primer-template (40 nm) was incubated with 0.04 nm of polκ in the absence of dNTP (0) or in the presence of a single dNTP (A, T, G, and C) or all of the four dNTPs (4). The reaction products were analyzed, as described in the legends to Fig. 1.
Figure 3
Figure 3
Bypass synthesis by polκΔC past abasic site. (A) Translesion synthesis by polκΔC. The template annealed with its complementary 13mer primer was incubated with polκΔC at three different concentrations (0.04, 0.4, and 4 nm from left to right) in the presence of four dNTPs. TT denotes the control nondamaged templates and the AP-T template was used for abasic site (AP) translesion synthesis. (B,C) The AP-T or AP-A template annealed with the complementary 16mer primer was reacted with 4 nm of polκΔC (D) Following the incubation with polκΔC (4 nm for the AP-T template and 40 nm for the AP-A template) for 15 min in the presence of four dNTPs, Klenow enzyme (exo+ 10 fmoles) was added to complete elongation to the end of the template. After a further 15-min incubation at 37°C, the replication products were analyzed. (E) A plausible model for bypass of abasic site by polκ. X denotes an abasic site.
Figure 4
Figure 4
Bypass synthesis by polκΔC past AAF-adduct. (A) The 30mer (AAF-A, 40 nm each) containing the AAF–adduct annealed with its complementary 13mer primer was incubated with three different concentrations of polκΔC (0.04, 0.4, and 4 nm from left to right) in the presence of four dNTPs. G indicates the control nondamaged template. (B) The 30mer containing the cisplatin–adduct annealed with its complementary 13mer primer was treated similarly. GG indicates the control nondamaged template. (C,D) The AAF-A or AAF-T template was annealed with the complementary 16mer primer and the primer-template was reacted with 4 nm of polκΔC. (E) Following the incubation with polκΔC for 15 min in the presence of four dNTPs, Klenow enzyme (exo+ 10 fmoles) was added. After a further 15-min incubation at 37°C, the replication products were analyzed.

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