5-Formylcytosine-induced DNA–peptide cross-links reduce transcription efficiency, but do not cause transcription errors in human cells

Ji, Shaofei; Park, Daeyoon; Kropachev, Konstantin; Kolbanovskiy, Marina; Fu, Iwen; Broyde, Suse; Essawy, Maram M.; Geacintov, Nicholas E.; Tretyakova, Natalia

doi:10.1074/jbc.ra119.009834

Cited by 19 publications

(21 citation statements)

References 35 publications

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“…Accumulation of these cross-links has been associated with aging, cancer, neurodegeneration, and Ruijs–Aalfs syndrome ( 2 , 3 , 4 , 5 , 6 ), and there has been considerable interest in both DNA–protein cross-links and proteases that can act on them ( 6 ). Several laboratories have shown that both reversible and irreversible DNA–protein cross-links can be formed under physiological conditions, that there are DNA-stimulated proteases that can act on these, and that DNA–protein cross-links can be bypassed and can miscode ( 2 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ). The number of cross-links in a cell has been estimated to be high ( 20 ) but the exact number is not known.…”

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confidence: 99%

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

Ghodke¹,

Gonzalez-Vasquez²,

Wang

et al. 2021

Journal of Biological Chemistry

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Unrepaired DNA–protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA–protein cross-links can be cleaved into DNA–peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide–DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant ( k cat / K m ) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.

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confidence: 99%

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

Ghodke¹,

Gonzalez-Vasquez²,

Wang

et al. 2021

Journal of Biological Chemistry

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“…A straightforward and precise way for the targeted incorporation of synthetic DNA lesions into plasmid-based vectors is ligation of short synthetic DNA strands into gapped circular DNA, which can be conveniently generated using sequence-specific nicking endonucleases [31,[52][53][54][55][56]. The use of nicking endonucleases for introducing modifications can be problematic in the protein coding regions of reporter genes, because conserved amino acid sequence restrains the possibilities for accommodation of the required recognition sites.…”

Section: Discussionmentioning

confidence: 99%

EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions

2020

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The sustainment of replication and transcription of damaged DNA is essential for cell survival under genotoxic stress; however, the damage tolerance of these key cellular functions comes at the expense of fidelity. Thus, translesion DNA synthesis (TLS) over damaged nucleotides is a major source of point mutations found in cancers; whereas erroneous bypass of damage by RNA polymerases may contribute to cancer and other diseases by driving accumulation of proteins with aberrant structure and function in a process termed “transcriptional mutagenesis” (TM). Here, we aimed at the generation of reporters suited for direct detection of miscoding capacities of defined types of DNA modifications during translesion DNA or RNA synthesis in human cells. We performed a systematic phenotypic screen of 25 non-synonymous base substitutions in a DNA sequence encoding a functionally important region of the enhanced green fluorescent protein (EGFP). This led to the identification of four loss-of-fluorescence mutants, in which any ulterior base substitution at the nucleotide affected by the primary mutation leads to the reversal to a functional EGFP. Finally, we incorporated highly mutagenic abasic DNA lesions at the positions of primary mutations and demonstrated a high sensitivity of detection of the mutagenic DNA TLS and TM in this system.

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“…72,80 Furthermore, both 5fC and 5caC induce a reduction in transcriptional efficiency-a counterintuitive effect considering the link between TET enzyme mediated demethylation and transcriptional activation. 57,81 Thus, transcriptional regulation by DNA and are mutually exclusive with TET2 mutations in AML. 133 When analyzing the cumulative incidences (…”

Section: The Significance Of 5mc Oxidation Derivativesmentioning

confidence: 99%

“…A mutagenic effect of 5fC was demonstrated prior to the discovery of the TET enzyme mechanism of formation, whereas 5caC was also shown to induce MMR protein activity 72,80 . Furthermore, both 5fC and 5caC induce a reduction in transcriptional efficiency—a counterintuitive effect considering the link between TET enzyme mediated demethylation and transcriptional activation 57,81 . Thus, transcriptional regulation by DNA (de)methylation/deamination should be considered, in all respects, a repurposing of DNA damage during evolution, by which chemical “lesions” have been redeployed as physiological elements of the epigenetic syntax, which again required the involvement of DNA repair enzymes like TDG to “check and balance” dioxygenase/deaminase activity in order to maintain genomic and epigenomic stability (Figure 3).…”

Section: The Significance Of 5mc Oxidation Derivativesmentioning

confidence: 99%

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Active DNA demethylation—The epigenetic gatekeeper of development, immunity, and cancer

2020

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DNA methylation is a critical process in the regulation of gene expression with dramatic effects in development and continually expanding roles in oncogenesis. 5-Methylcytosine was once considered to be an inherited and stably repressive epigenetic mark, which can be only removed by passive dilution during multiple rounds of DNA replication. However, in the past two decades, physiologically controlled DNA demethylation and deamination processes have been identified, thereby revealing the function of cytosine methylation as a highly regulated and complex state-not simply a static, inherited signature or binary onoff switch. Alongside these fundamental discoveries, clinical studies over the past decade have revealed the dramatic consequences of aberrant DNA demethylation. In this review we discuss DNA demethylation and deamination in the context of 5-methylcytosine as critical processes for physiological and physiopathological transitions within three statesdevelopment, immune maturation, and oncogenic transformation; and we describe the expanding role of DNA demethylating drugs as therapeutic agents in cancer.

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5-Formylcytosine-induced DNA–peptide cross-links reduce transcription efficiency, but do not cause transcription errors in human cells

Cited by 19 publications

References 35 publications

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions

Active DNA demethylation—The epigenetic gatekeeper of development, immunity, and cancer

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