Methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCG→TTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3→5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

Repair and DNA Polymerase Bypass of Clickable Pyrimidine Nucleotides.

Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase ß. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.

Base Excision DNA Repair in Plants: Arabidopsis and Beyond.

Base excision DNA repair (BER) is a key pathway safeguarding the genome of all living organisms from damage caused by both intrinsic and environmental factors. Most present knowledge about BER comes from studies of human cells, E. coli, and yeast. Plants may be under an even heavier DNA damage threat from abiotic stress, reactive oxygen species leaking from the photosynthetic system, and reactive secondary metabolites. In general, BER in plant species is similar to that in humans and model organisms, but several important details are specific to plants. Here, we review the current state of knowledge about BER in plants, with special attention paid to its unique features, such as the existence of active epigenetic demethylation based on the BER machinery, the unexplained diversity of alkylation damage repair enzymes, and the differences in the processing of abasic sites that appear either spontaneously or are generated as BER intermediates. Understanding the biochemistry of plant DNA repair, especially in species other than the Arabidopsis model, is important for future efforts to develop new crop varieties.

Photoinduced inhibition of DNA repair enzymes and the possible mechanism of photochemical transformations of the ruthenium nitrosyl complex [RuNO(ß-Pic)2(NO2)2OH].

In this work we have demonstrated that the ruthenium nitrosyl complex [RuNO(ß-Pic)2(NO2)2OH] is suitable for investigation of the inactivation of DNA repair enzymes in vitro. Photoinduced inhibition of DNA glycosylases such as E. coli Endo III, plant NtROS1, mammalian mNEIL1 and hNEIL2 occurs to an extent of ≥90% after irradiation with the ruthenium complex. The photophysical and photochemical processes of [RuNO(ß-Pic)2(NO2)2OH] were investigated using stationary and time-resolved spectroscopy, and mass spectrometry. A possible mechanism of the photo-processes was proposed from the combined spectroscopic study and DTF calculations, which reveal that the photolysis is multistage. The primary and secondary photolysis stages are the photo-induced cleavage of the Ru-NO bond with the formation of a free nitric oxide and RuIII complex followed by ligand exchange with solvent. For E. coli Endo III, covalent interaction with the photolysis product was confirmed by UV-vis and mass spectrometric methods.

Delayed effects of accelerated heavy ions on the induction of HPRT mutations in V79 hamster cells.

Fundamental research on the harmful effects of ionizing radiation on living cells continues to be of great interest. Recently, priority has been given to the study of high-charge and high-energy (HZE) ions that comprise a substantial part of the galactic cosmic ray (GCR) spectra that would be encountered during long-term space flights. Moreover, predictions of the delayed genetic effects of high linear energy transfer (LET) exposure is becoming more important as heavy ion therapy use is increasing. This work focuses mainly on the basic research on the delayed effects of HZE ions on V79 Chinese hamster cells, with emphasis on the induction of HPRT mutations after prolonged expression times (ET). The research was conducted under various irradiation conditions with accelerated ions 18O (E=35.2MeV/n), 20Ne (E=47.7MeV/n and 51.8MeV/n), and 11B (E=32.4MeV/n), with LET in the range from 49 to 149 keV/µm and with 60Co γ-rays. The HPRT mutant fractions (MF) were detected in irradiated cells in regular intervals during every cell culture recultivation (every 3days) up to approximately 40days (70-80 generations) after irradiation. The MF maximum was reached at different ET depending on ionizing radiation characteristics. The position of the maximum was shifting towards longer ET with increasing LET. We speculate that the delayed mutations are created de novo and that they are the manifestation of genomic instability. Although the exact mechanisms involved in genomic instability initiation are yet to be identified, we hypothesize that differences in induction of delayed mutations by radiations with various LET values are related to variations in energy deposition along the particle track. A dose dependence of mutation yield is discussed as well.