DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8–2′-deoxyguanosine adduct of the dietary mutagen IQ in human cells

Self Cite

DNA-protein cross-links are formed upon exposure of cellular DNA to various agents, including antitumor drugs, UV light, transition metals, and reactive oxygen species. They are thought to contribute to cancer, aging, and neurodegenerative diseases. It has been proposed that DNA-protein cross-links formed in cells are subject to proteolytic degradation to the corresponding DNA-peptide cross-links (DpCs). To investigate the effects of DpCs on DNA replication, we have constructed plasmid DNA containing a 10-mer Myc peptide covalently linked to C7 of 7-deaza-dG, a hydrolytically stable mimic of N7-dG lesions. Following transfection in human embryonic kidney cells (HEK 293T), progeny plasmids were recovered and sequenced. Translesion synthesis (TLS) past DpC was 76% compared to unmodified control. The DpC induced 20% targeted G→A and G→T plus 15% semi-targeted mutations, notably at a guanine (G5) five bases 3’ to the lesion site. Proteolytic digestion of the DpC reduced the mutation frequency considerably, indicating that the covalently attached 10-mer peptide was responsible for the observed mutations. TLS efficiency and targeted mutations were reduced upon siRNA knockdown of pol η, pol κ, or pol ζ, indicating that they participate in error-prone bypass of the DpC lesion. However, the semi-targeted mutation at G5 was only reduced upon knockdown of pol ζ, suggesting its critical role in this type of mutations. Our results indicate that DpCs formed at the N7 position of guanine can induce both targeted and semi-targeted mutations in human cells and that the TLS polymerases play a critical role in their error-prone bypass.

Section: Resultsmentioning

confidence: 99%

“…30 The mixed DNA was used to transfect HEK 293T cells and processed as described above. Oligonucleotide probes for the complementary sequences for both the wild type and the mutant plasmid were used to analyze the progeny.…”

Section: Methodsmentioning

confidence: 99%

Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases

Pande

Mukherjee³

et al. 2016

Self Cite

“…Site-specific adduct-containing pMS2 vectors with neomycin and ampicillin resistance genes were constructed in a manner similar to the construction of the dG-C8-IQ-containing vectors. 42 Transfection of 50 ng of each construct was performed in HEK293T cells, after they were grown to ∼90% confluency, using 6 μL of Lipofectamine cationic lipid reagent (Invitrogen, Carlsbad, CA). Subsequently, the cells were grown at 37 °C in 5% CO 2 for 48 h, and the plasmid DNA was isolated and purified.…”

Section: Methodsmentioning

confidence: 99%

“…32 P-Labeling, annealing, and extension reactions of the primers on the unmodified or the dG- N 2 -IQ modified template by Rev1 were carried out in the presence of dCTPs in a manner similar to that in ref ( 42 ). Details are provided in the SI .…”

Section: Methodsmentioning

confidence: 99%

Translesion Synthesis of the N²-2′-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error-Free Replication by DNA Polymerase κ and Mutagenic Bypass by DNA Polymerases η, ζ, and Rev1

Bose

Millsap

DeLeon

et al. 2016

Self Cite

Translesion synthesis (TLS) of the N2-2′-deoxyguanosine (dG-N2-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5′-CG1G2CG3CC-3′). TLS efficiency was 38%, 29%, and 25% for the dG-N2-IQ located at G1, G2, and G3, respectively, which suggests that dG-N2-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8–35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N2-IQ bypass. Mutation frequencies (MFs) of dG-N2-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 (26220181Nucleic Acids Res.20154383408351). The major type of mutation induced by dG-N2-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N2-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N2-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.

“…135 MF was the highest (50%) when the adduct was placed at G 3 , compared to 18% and 24% MF when the adduct was located at G 1 and G 2 , respectively, inducing mainly G → T transversions at each site. MF of dG-C8-IQ was reduced in varying degrees upon siRNA knockdown of pol κ, pol ι-, pol ζ-, or Rev1-knockdown cells (Table 2), indicating that these pols are involved in error-prone synthesis of this adduct.…”

Section: Tls Of Bulky Dg Lesionsmentioning

confidence: 91%

Translesion Synthesis of 2′-Deoxyguanosine Lesions by Eukaryotic DNA Polymerases

Basu

Pande

Bose

2016

Self Cite

With the discovery of translesion synthesis DNA polymerases, great strides have been made in the last two decades in understanding the mode of replication of various DNA lesions in prokaryotes and eukaryotes. A database search indicated that approximately 2000 articles on this topic have been published in this period. This includes research involving genetic and structural studies as well as in vitro experiments using purified DNA polymerases and accessory proteins. It is a daunting task to comprehend this exciting and rapidly emerging area of research. Even so, as the majority of DNA damage occurs at 2′-deoxyguanosine residues, this perspective attempts to summarize a subset of this field, focusing on the most relevant eukaryotic DNA polymerases responsible for their bypass.