Identification of DNA lesions using a third base pair for amplification and nanopore sequencing

Riedl, Jan; Ding, Yun; Fleming, Aaron M.; Burrows, Cynthia J.

doi:10.1038/ncomms9807

Cited by 76 publications

(111 citation statements)

References 64 publications

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“…Insertion of the PQS and nicking endonuclease recognition sequences was achieved using restriction-free cloning, followed by transformation to competent Escherichia coli and isolation by miniprep kit (Qiagen), as described previously (38). The site-specific modifications were synthesized into short oligomers with the sequence between the two nicking endonuclease sites and they were inserted into the plasmid via literature methods (38,39). Confirmation that the DNA modifications were introduced into the plasmid was achieved using a protocol established in the C.J.B.…”

Section: Methodsmentioning

confidence: 99%

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

Fleming

Ding

Burrows

2017

Proc. Natl. Acad. Sci. U.S.A.

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278

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Reactive oxygen species (ROS) have emerged as important cellularsignaling agents for cellular survival. Herein, we demonstrate that ROS-mediated oxidation of DNA to yield 8-oxo-7,8-dihydroguanine (OG) in gene promoters is a signaling agent for gene activation. Enhanced gene expression occurs when OG is formed in guanine-rich, potential G-quadruplex-forming sequences (PQS) in promoter-coding strands, initiating base excision repair (BER) by 8-oxoguanine DNA glycosylase (OGG1), yielding an abasic site (AP). The AP enables melting of the duplex to unmask the PQS, adopting a G-quadruplex fold in which apurinic/apyrimidinic endonuclease 1 (APE1) binds, but inefficiently cleaves, the AP for activation of vascular endothelial growth factor (VEGF) or endonuclease III-like protein 1 (NTHL1) genes. These details were mapped via synthesis of OG and AP analogs at singlenucleotide precision within the promoter of a luciferase reporter system. The reporters were analyzed in human and mouse cells while selectively knocking out or down critical BER proteins to identify the impact on luciferase expression. Identification of the oxidatively modified DNA base OG to guide BER activity in a gene promoter and impact cellular phenotype ascribes an epigenetic role to OG.

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Section: Methodsmentioning

confidence: 99%

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

Fleming

Ding

Burrows

2017

Proc. Natl. Acad. Sci. U.S.A.

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278

432

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“…Many laboratories have developed antibody-based OG sequencing that provides a low resolution sequence map of OG (~10–1000 kbp) [53, 54]. Recently, our laboratory developed an OG sequencing approach with single-nucleotide resolution implemented on plasmid DNA [55], in addition to an OG sequencing method with ~0.15-kbp resolution (i.e., OG-Seq) that was implemented on the mouse genome [56]. Expansion of sequencing studies, ideally at single-nucleotide resolution, to different cell types under a variety of conditions (i.e., different stressors or cell states) will be essential to the determination of the sequences in which OG is preferentially formed, whether these sequences and regions change based on cellular conditions, and whether they are gene regulatory regions.…”

Section: How Does Og Fit Into the Epigenetic Landscape?mentioning

confidence: 99%

8-Oxo-7,8-dihydroguanine, friend and foe: Epigenetic-like regulator versus initiator of mutagenesis

2017

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A high flux of reactive oxygen species during oxidative stress results in oxidative modification of cellular components including DNA. Oxidative DNA “damage” to the heterocyclic bases is considered deleterious because polymerases may incorrectly read the modifications causing mutations. A prominent member in this class is the oxidized guanine base 8-oxo-7,8-dihydroguanine (OG) that is moderately mutagenic effecting G→T transversion mutations. Recent reports have identified that formation of OG in G-rich regulatory elements in the promoters of the VEGF, TNFα, and SIRT1 genes can increase transcription via activation of the base excision repair (BER) pathway. Work in our laboratory with the G-rich sequence in the promoter of VEGF concluded that BER drives a shift in structure to a G-quadruplex conformation leading to gene activation in mammalian cells. More specifically, removal of OG from the duplex context by 8-oxoguanine glycosylase 1 (OGG1) produces an abasic site (AP) that destabilizes the duplex, shifting the equilibrium toward the G-quadruplex fold because of preferential extrusion of the AP into a loop. The AP is bound but inefficiently cleaved by apurinic/apyrimidinic endoDNase I (APE1) that likely allows recruitment of activating transcription factors for gene induction. The ability of OG to induce transcription ascribes a regulatory or epigenetic-like role for this oxidatively modified base. We compare OG to the 5-methylcytosine (5mC) epigenetic pathway including its oxidized derivatives, some of which poise genes for transcription while also being substrates for BER. The mutagenic potential of OG to induce only ~one-third the number of mutations (G→T) compared to deamination of 5mC producing C→T mutations is described. These comparisons blur the line between friendly epigenetic base modifications and those that are foes, i.e. DNA “damage,” causing genetic mutations.

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“…Other sequencing-based methods developed over the last years allow direct sequencing of different lesion types, either by the analysis of alterations in the kinetics of DNA polymerases with single-molecule real-time (SMRT) DNA sequencing, 26 or by the introduction and sequencing of an additional unnatural base pair at the lesion sites that can be directly sequenced with the α-hemolysin nanopore system 27 . However, considering the high error rates of both these methods and the technical challenge when measuring higher lesion numbers, to date they provide a tool for the direct study of several DNA lesions, but are not well suited for the analysis of ultra-rare sequence variants.…”

Section: Introductionmentioning

confidence: 99%

Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

Arbeithuber

Makova

Tiemann‐Boege

2016

DNA Res

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The need in cancer research or evolutionary biology to detect rare mutations or variants present at very low frequencies (<10−5) poses an increasing demand on lowering the detection limits of available methods. Here we demonstrated that amplifiable DNA lesions introduce important error sources in ultrasensitive technologies such as single molecule PCR (smPCR) applications (e.g. droplet-digital PCR), or next-generation sequencing (NGS) based methods. Using templates with known amplifiable lesions (8-oxoguanine, deaminated 5-methylcytosine, uracil, and DNA heteroduplexes), we assessed with smPCR and duplex sequencing that templates with these lesions were amplified very efficiently by proofreading polymerases (except uracil), leading to G->T, and to a lesser extent, to unreported G->C substitutions at 8-oxoguanine lesions, and C->T transitions in amplified uracil containing templates. Long heat incubations common in many DNA extraction protocols significantly increased the number of G->T substitutions. Moreover, in ∼50-80% smPCR reactions we observed the random amplification preference of only one of both DNA strands explaining the known ‘PCR jackpot effect’, with the result that a lesion became indistinguishable from a true mutation or variant. Finally, we showed that artifactual mutations derived from uracil and 8-oxoguanine could be significantly reduced by DNA repair enzymes.

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Identification of DNA lesions using a third base pair for amplification and nanopore sequencing

Cited by 76 publications

References 64 publications

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

8-Oxo-7,8-dihydroguanine, friend and foe: Epigenetic-like regulator versus initiator of mutagenesis

Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

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