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. 2015 Sep 30;43(17):8340-51.
doi: 10.1093/nar/gkv750. Epub 2015 Jul 28.

DNA Polymerases κ and ζ Cooperatively Perform Mutagenic Translesion Synthesis of the C8-2'-deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells

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DNA Polymerases κ and ζ Cooperatively Perform Mutagenic Translesion Synthesis of the C8-2'-deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells

Arindam Bose et al. Nucleic Acids Res. .
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Abstract

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8-2'-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G1-, G2- or G3-positions of the NarI site (5'-G1G2CG3CC-3') were replicated in HEK293T cells. Fifty percent of the progeny from the G3 construct were mutants, largely G→T, compared to 18% and 24% from the G1 and G2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38-67% upon siRNA knockdown of pol κ, whereas it was increased by 10-24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G1 and G3 constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G2 construct. In vitro TLS using yeast pol ζ showed that it can extend G3*:A pair more efficiently than G3*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.

Figures

Figure 1.
Figure 1.
Metabolic activation and the major DNA adduct formation by IQ.
Scheme 1.
Scheme 1.
General protocol for construction of dG-C8-IQ-containing pMS2 plasmid and its replication in HEK293T cells. Mutational analyses of the progeny were carried out by oligonucleotide hybridization. The 15-mer left (LP) and 15-mer right (RP) probes were used to select plasmids containing the correct insert, and transformants that did not hybridize with both the left and right probes were omitted. A probe containing the complementary 14-mer wild type sequence (WTP) was used to analyze the progeny plasmids. An example of a wild type progeny is shown by the green circle. Any transformant that hybridized with the left and right probes but failed to hybridize with the 14-mer wild type probe (as shown by the red circle) was considered a putative mutant and subjected to DNA sequence analysis.
Figure 2.
Figure 2.
Effects of siRNA knockdowns of TLS pols on the extent of replicative bypass of dG-C8-IQ. Percent TLS in various pol knockdowns was measured relative to an internal control in which a different 12-mer oligonucleotide was inserted (i.e. 5′-GTGCGTGTTTGT-3′ in place of 5′-CTCG1G2CG3CCATC-3′) into the gapped plasmid in a manner similar to the construction of the dG-C8-IQ (or control) construct. The data represent the means and standard deviations of results from two independent experiments. HEK293T cells were treated with negative control (NC) siRNA (WT), whereas the other single or double pol(s) knockdowns are indicated above the bar. TLS result from each knockdown experiment was considered statistically significant (P < 0.05) compared to that from HEK 293T cells treated with NC siRNA. The P-value of %TLS for each knockdown was calculated by using two-tailed, unpaired Student's t-test.
Figure 3.
Figure 3.
Mutational frequency of dG-C8-IQ in the progeny from the G1, G2 and G3 constructs in HEK293T cells also transfected with NC siRNA (WT) or siRNA for single, double or triple pol(s) knockdowns (as indicated above the bar) is shown. The data represent the average of two independent experiments (presented in Supplementary Table S1 A–J in the SI).
Figure 4.
Figure 4.
The types and frequencies of mutations induced by dG-C8-IQ in the progeny from the G1, G2 and G3 constructs in HEK293T cells also transfected with NC siRNA (293T) or siRNA for single pol knockdowns are shown in a pie chart. O represents other mutations. The data represent the average of two independent experiments (presented in Supplementary Table S1 A–F in the SI).
Figure 5.
Figure 5.
The types and frequencies of mutations induced by dG-C8-IQ in the progeny from the G1, G2 and G3 constructs in HEK293T cells also transfected with NC siRNA (293T) or siRNA for double or triple pol(s) knockdowns are shown in a pie chart. D and O, respectively, represent targeted deletions and other mutations. The data represent the average of two independent experiments (presented in Supplementary Table S1 G–J in the SI).
Figure 6.
Figure 6.
In vitro insertion and extension assay of the dG3-C8-IQ adduct by ypol ζ. (A) Insertion of dCTP opposite a control unmodified dG (left) and reaction of the dG-C8-IQ modified oligonucleotide in the presence of all four dNTPs. (B) Extension of the dG3-C8-IQ adduct when paired with C or A after 5 (left) and 24 (right) h.
Figure 7.
Figure 7.
(A) Extension past a dG3-C8-IQ:N pair (N = C, A and T; 10 nM) by hpol κ after 5 h at 37°C in the presence of all four dNTPs (100 μM). (B) Primer extension of a G:C primer template terminus (+1 position) and dG3-C8-IQ:N pair (N = C, A and T; 0-position) by hpol κ after 5 h at 37°C in the presence of all four dNTPs (100 μM). The DNA concentration was 10 nM.

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