Enzymatic Hydroxylation and Excision of Extended 5-Methylcytosine Analogues

Tomkuvienė, Miglė; Ikasalaitė, Diana; Slyvka, Anton; Rukšėnaitė, Audronė; Ravichandran, Mirunalini; Jurkowski, Tomasz P.; Bochtler, Matthias; Klimašauskas, Saulius

doi:10.1016/j.jmb.2020.10.011

Cited by 7 publications

(2 citation statements)

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“…Purification was carried out similarly as described in ref. 48 . In brief, harvested cells were washed once with 1X STE (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA) buffer, the pellet was then resuspended in sonication/wash buffer (50 mM HEPES pH 6.8, 500 mM NaCl, 1 mM DTT, 35 mM imidazole, 10 mM alpha-ketoglutarate, and 10% glycerol) supplemented with Protease Inhibitor cocktail (final 300 µM AEBSF-HCl, 3 µM Pepstatin A, 0.12 µM Aprotinin, 15 µM Bestatin, 4.5 µM E-64, 6.7 µM Leupeptin) and 0.2 mM PMSF (Sigma) and lysed by sonication (15 cycles, 15 s with 30% power, 45 s off).…”

Section: Methodsmentioning

confidence: 99%

Flanking sequences influence the activity of TET1 and TET2 methylcytosine dioxygenases and affect genomic 5hmC patterns

et al. 2022

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TET dioxygenases convert 5-methylcytosine (5mC) preferentially in a CpG context into 5-hydroxymethylcytosine (5hmC) and higher oxidized forms, thereby initiating DNA demethylation, but details regarding the effects of the DNA sequences flanking the target 5mC site on TET activity are unknown. We investigated oxidation of libraries of DNA substrates containing one 5mC or 5hmC residue in randomized sequence context using single molecule readout of oxidation activity and sequence and show pronounced 20 and 70-fold flanking sequence effects on the catalytic activities of TET1 and TET2, respectively. Flanking sequence preferences were similar for TET1 and TET2 and also for 5mC and 5hmC substrates. Enhanced flanking sequence preferences were observed at non-CpG sites together with profound effects of flanking sequences on the specificity of TET2. TET flanking sequence preferences are reflected in genome-wide and local patterns of 5hmC and DNA demethylation in human and mouse cells indicating that they influence genomic DNA modification patterns in combination with locus specific targeting of TET enzymes.

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Section: Methodsmentioning

confidence: 99%

Flanking sequences influence the activity of TET1 and TET2 methylcytosine dioxygenases and affect genomic 5hmC patterns

et al. 2022

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“…HPLC-MS/MS analysis was performed as described by Tomkuviene et al [ 33 ] with modifications. DNA (2–4 µg per sample) hydrolyzates were analyzed on an integrated HPLC/ESI-MS/MS system (Agilent 1290 Infinity/ 6410 Triple Quad) equipped with a Supelco DiscoveryHS C18 column (75 × 2.1 mm, 3 μm) by elution with a linear gradient of solvents A (0.0075% formic acid in water) and B (0.0075% formic acid in acetonitrile) at a flow of 0.3 ml/min at 30 °C as follows: 0–6 min, 0% B; 6–18 min, 10% B; 18–20 min, 100% B.…”

Section: Methodsmentioning

confidence: 99%

FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine

et al. 2022

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Fanconi anemia (FA) is a rare hereditary disorder caused by mutations in any one of the FANC genes. FA cells are mainly characterized by extreme hypersensitivity to interstrand crosslink (ICL) agents. Additionally, the FA proteins play a crucial role in concert with homologous recombination (HR) factors to protect stalled replication forks. Here, we report that the 5-methyl-2’-deoxycytidine (5mdC) demethylation (pathway) intermediate 5-hydroxymethyl-2’-deoxycytidine (5hmdC) and its deamination product 5-hydroxymethyl-2’-deoxyuridine (5hmdU) elicit a DNA damage response, chromosome aberrations, replication fork impairment and cell viability loss in the absence of FANCD2. Interestingly, replication fork instability by 5hmdC or 5hmdU was associated to the presence of Poly(ADP-ribose) polymerase 1 (PARP1) on chromatin, being both phenotypes exacerbated by olaparib treatment. Remarkably, Parp1−/− cells did not show any replication fork defects or sensitivity to 5hmdC or 5hmdU, suggesting that retained PARP1 at base excision repair (BER) intermediates accounts for the observed replication fork defects upon 5hmdC or 5hmdU incorporation in the absence of FANCD2. We therefore conclude that 5hmdC is deaminated in vivo to 5hmdU, whose fixation by PARP1 during BER, hinders replication fork progression and contributes to genomic instability in FA cells.

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