Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion

Abstract

REV1 functions in the DNA polymerase zeta mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3'-->5' proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19-27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N2-dG, (-)-trans-anti-benzo[a]pyrene-N2-dG and 1,N6-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6-4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase kappa, bypass of the trans-anti-benzo[a]pyrene-N2-dG adducts and the 1,N6-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass.

Figure 1

DNA polymerase activity of human REV1 on a repeating template G sequence. (A) The purified human REV1 protein (400 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) DNA polymerase assays were performed in the presence of a single dATP (A), dCTP (C), dTTP (T), dGTP (G) or all four dNTPs (N₄) at 30°C for 30 min, using 7 ng (50 fmol) of purified human REV1 and 50 fmol of the indicated template containing a 5′-³²P-labeled 20mer primer. DNA size markers in nucleotides are indicated on the left. Quantitation of extended primers is shown below the gel.

Figure 2

Proofreading exonuclease assays of human REV1 protein. A 15mer 5′-³²P-labeled primer was annealed to the indicated DNA template, forming an A–C (template–primer) mismatch at the primer 3′ end. The DNA substrate was incubated with the purified Klenow fragment of E.coli DNA polymerase I (1 U) (lane 2), purified human REV1 protein (10 ng, 72 fmol) (lane 3) or without any protein (lane 1) at 37°C. The reaction was 2 min for the Klenow fragment and 30 min for human REV1. DNA size markers in nucleotides are indicated on the left.

Figure 3

Activity of human REV1 opposite various template bases. (A) Templates used for DNA polymerase assays. A 17mer primer was labeled with ³²P at its 5′ end as indicated by an asterisk and annealed separately to each DNA template. (B) Standard DNA polymerase assays were performed with 50 fmol DNA substrate and 10 ng (72 fmol) of human REV1 in the presence of a single dATP (A), dCTP (C), dTTP (T), dGTP (G) or all four dNTPs (N₄) as indicated. DNA size markers in nucleotides are indicated on the sides.

Figure 4

Kinetic analysis of nucleotide insertion by human REV1. Standard DNA polymerase assays were performed at 30°C for 2 min, using 2 ng (14 fmol) of purified human REV1 and 50 fmol of DNA templates containing a ³²P-labeled 17mer primer. Increasing concentrations of a single dNTP were used in the assay. The reaction products were separated by electrophoresis on a 15% denaturing polyacrylamide gel and visualized by autoradiography. After quantifying extended primers as shown below the gels, the kinetic V_max and K_m values were obtained as described in Materials and Methods. Autoradiograms of dCMP insertion opposite templates C and G, respectively, are shown. DNA templates used are shown in Figure 3A. DNA size markers in nucleotides are indicated on the sides.

Figure 5

Nucleotide insertion by human REV1 opposite various DNA lesions. DNA primers were labeled with ³²P (asterisks) at their 5′ ends and separately annealed right before a template lesion as indicated. Standard DNA polymerase assays with the damaged DNA template (50 fmol) were performed in the presence of a single dATP (A), dCTP (C), dTTP (T), dGTP (G) or all four dNTPs (N₄) at 30°C for 10 min, using 10 ng (72 fmol) (A, B and D) or 40 ng (290 fmol) (C) of purified human REV1. (A) Template containing an 8-oxoguanine and a 17mer primer. (B) Templates containing a (+)-trans-anti-BPDE-N²-dG adduct or (–)-trans-anti-BPDE-N²-dG adduct, and a 19mer primer. (C) Template containing an AAF-guanine adduct and an 18mer primer. (D) Template containing a 1,N⁶-ethenoadenine and a 20mer primer. DNA size markers in nucleotides are indicated on the left or on the sides.

Figure 6

DNA lesion bypass by the combined activities of human REV1 and Polκ. Standard DNA polymerase assays with the indicated DNA templates (50 fmol) were performed in the presence of all four dNTPs, using 10 ng (72 fmol) (A) or 5 ng (36 fmol) (B) of purified human REV1. After 10 min reaction at 30°C, 5 ng (50 fmol) of purified human Polκ was then added to the reaction and the incubation continued for another 10 min. (A) Bypass of the 1,N⁶-ethenoadenine lesion. (B) Bypass of the (+)- and (–)-trans-anti-BPDE-N²-dG adducts. Lanes 1 and 3, reactions with REV1 alone for 20 min at 30°C; P, the primer. DNA size markers in nucleotides are indicated on the right.

Figure 7

Response of human REV1 to a template TT dimer and TT (6–4) photoproduct. Two primers were labeled with ³²P at their 5′ ends and separately annealed to a template containing a site-specific TT dimer or TT (6–4) photoproduct as shown. The 3′ T primer (15mer) and the 5′ T primer (16mer) were annealed right before the 3′ T and the 5′ T of the lesion, respectively. Standard DNA polymerase assays were performed with 50 fmol of the damaged DNA template and 40 ng (290 fmol) of human REV1 in the presence of all four dNTPs. (A) DNA polymerase assays with substrates containing the 3′ T primer. (B) DNA polymerase assays with substrates containing the 5′ T primer. Lane 1, reaction with the TT dimer template without human REV1 protein. DNA size markers in nucleotides are indicated on the left.