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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 β. 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. [ABSTRACT FROM AUTHOR]

The DNA building blocks 2′-deoxynucleotides are enantiomeric, with their natural β-D-configuration dictated by the sugar moiety. Their synthetic β-L-enantiomers (βLdNs) can be used to obtain L-DNA, which, when fully substituted, is resistant to nucleases and is finding use in many biosensing and nanotechnology applications. However, much less is known about the enzymatic recognition and processing of individual βLdNs embedded in D-DNA. Here, we address the template properties of βLdNs for several DNA polymerases and the ability of base excision repair enzymes to remove these modifications from DNA. The Klenow fragment was fully blocked by βLdNs, whereas DNA polymerase κ bypassed them in an error-free manner. Phage RB69 DNA polymerase and DNA polymerase β treated βLdNs as non-instructive but the latter enzyme shifted towards error-free incorporation on a gapped DNA substrate. DNA glycosylases and AP endonucleases did not process βLdNs. DNA glycosylases sensitive to the base opposite their cognate lesions also did not recognize βLdNs as a correct pairing partner. Nevertheless, when placed in a reporter plasmid, pyrimidine βLdNs were resistant to repair in human cells, whereas purine βLdNs appear to be partly repaired. Overall, βLdNs are unique modifications that are mostly non-instructive but have dual non-instructive/instructive properties in special cases. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions generated both by spontaneous base loss and as intermediates of base excision DNA repair. In human cells, they are normally repaired by an essential AP endonuclease, APE1, encoded by the APEX1 gene. Other enzymes can cleave AP sites by either hydrolysis or β-elimination in vitro, but it is not clear whether they provide the second line of defense in living cells. Here, we studied AP site repairs in APEX1 knockout derivatives of HEK293FT cells using a reporter system based on transcriptional mutagenesis in the enhanced green fluorescent protein gene. Despite an apparent lack of AP site-processing activity in vitro, the cells efficiently repaired the tetrahydrofuran AP site analog resistant to β-elimination. This ability persisted even when the second AP endonuclease homolog, APE2, was also knocked out. Moreover, APEX1 null cells were able to repair uracil, a DNA lesion that is removed via the formation of an AP site. If AP site hydrolysis was chemically blocked, the uracil repair required the presence of NTHL1, an enzyme that catalyzes β-elimination. Our results suggest that human cells possess at least two back-up AP site repair pathways, one of which is NTHL1-dependent. [ABSTRACT FROM AUTHOR]

Oxidative DNA lesions, constantly generated by both endogenous and environmentally induced reactive oxygen species, are removed via the base excision repair pathway. In bacteria, Fpg and Nei DNA glycosylases, belonging to the helix–two-turn–helix (H2TH) structural superfamily, remove oxidised purines and pyrimidines, respectively. Interestingly, the human H2TH family glycosylases, NEIL1, NEIL2 and NEIL3, have been reported to prefer oxidative lesions in DNA bubbles or single-stranded DNA. It had been hypothesised that NEIL2 might be involved in the repair of lesions in transcription bubbles; however, bubble-like structures may appear in other cellular contexts such as displacement loops (D-loops) associated with transcription, recombination or telomere maintenance. The activities of bacterial Fpg and Nei on bubble substrates were not addressed. Also, it is not known whether H2TH enzymes process bubbles containing the third DNA or RNA strand, and how the bubble length and position of the lesion within a bubble affect the excision. We have investigated the removal of 8-oxoguanine (8-oxoG) and 5,6-dihydrouracil (DHU) by Escherichia coli Fpg and Nei and human NEIL1 and NEIL2 from single-strand oligonucleotides, perfect duplexes, bubbles with different numbers of unpaired bases (6–30), bubbles containing the lesion in different positions and D-loops with the third strand made of DNA or RNA. Fpg, NEIL1 and NEIL2 efficiently excised lesions located within bubbles, with NEIL1 and NEIL2 being specific for DHU, and Fpg removing both 8-oxoG and DHU. Nei, in contrast, was significantly active only on DHU located in double-stranded DNA. Fpg and NEIL1 also tolerated the presence of the third strand of either DNA or RNA in D-loops if the lesion was in the single-stranded part, and Fpg, Nei and NEIL1 excised lesions from the double-stranded DNA part of D-loops. The presence of an additional unpaired 5′-tail of DNA or RNA did not affect the activity. No significant position preference for lesions in a 12-mer bubble was found. Overall, the activities of Fpg, NEIL1 and NEIL2 on these non-canonical substrates are consistent with the possibility that these enzymes may participate in the repair in structures arising during transcription or homologous recombination. [ABSTRACT FROM AUTHOR]

8-Oxoguanine (8-oxoG), a mutagenic DNA lesion generated under oxidative stress, differs from its precursor guanine by only two substitutions (O8 and H7). Human 8-oxoguanine glycosylase 1 (OGG1) can locate and remove 8-oxoG through extrusion and excision. To date, it remains unclear how OGG1 efficiently distinguishes 8-oxoG from a large excess of undamaged DNA bases. We recently showed that formamidopyrimidine-DNA glycosylase (Fpg), a bacterial functional analog of OGG1, can selectively facilitate eversion of oxoG by stabilizing several intermediate states, and it is intriguing whether OGG1 also employs a similar mechanism in lesion recognition. Here, we use molecular dynamics simulations to explore the mechanism by which OGG1 discriminates between 8-oxoG and guanine along the base-eversion pathway. The MD results suggest an important role for kinking of the DNA by the glycosylase, which positions DNA phosphates in a way that assists lesion recognition during base eversion. The computational predictions were validated through experimental enzyme assays on phosphorothioate substrate analogs. Our simulations suggest that OGG1 distinguishes between 8-oxoG and G using their chemical dissimilarities not only at the active site but also at earlier stages during base eversion, and this mechanism is at least partially conserved in Fpg despite a lack of structural homology. The similarity also suggests that lesion recognition through multiple gating steps may be a common theme in DNA repair. Our results provide new insight into how enzymes can exploit kinetics and DNA conformational changes to probe the chemical modifications present in DNA lesions. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions generated both by spontaneous base loss and as intermediates of base excision DNA repair. In human cells, they are normally repaired by an essential AP endonuclease, APE1, encoded by the APEX1 gene. Other enzymes can cleave AP sites by either hydrolysis or β-elimination in vitro, but it is not clear whether they provide the second line of defense in living cells. Here, we studied AP site repairs in APEX1 knockout derivatives of HEK293FT cells using a reporter system based on transcriptional mutagenesis in the enhanced green fluorescent protein gene. Despite an apparent lack of AP site-processing activity in vitro, the cells efficiently repaired the tetrahydrofuran AP site analog resistant to β-elimination. This ability persisted even when the second AP endonuclease homolog, APE2, was also knocked out. Moreover, APEX1 null cells were able to repair uracil, a DNA lesion that is removed via the formation of an AP site. If AP site hydrolysis was chemically blocked, the uracil repair required the presence of NTHL1, an enzyme that catalyzes β-elimination. Our results suggest that human cells possess at least two back-up AP site repair pathways, one of which is NTHL1-dependent.

In contrast to proteins recognizing small-molecule ligands, DNA-dependent enzymes cannot rely solely on interactions in the substrate-binding centre to achieve their exquisite specificity. It is widely believed that substrate recognition by such enzymes involves a series of conformational changes in the enzyme-DNA complex with sequential gates favoring cognate DNA and rejecting nonsubstrates. However, direct evidence for such mechanism is limited to a few systems. We report that discrimination between the oxidative DNA lesion, 8-oxoguanine (oxoG) and its normal counterpart, guanine, by the repair enzyme, formamidopyrimidine-DNA glycosylase (Fpg), likely involves multiple gates. Fpg uses an aromatic wedge to open the Watson-Crick base pair and everts the lesion into its active site. We used molecular dynamics simulations to explore the eversion free energy landscapes of oxoG and G by Fpg, focusing on structural and energetic details of oxoG recognition. The resulting energy profiles, supported by biochemical analysis of site-directed mutants disturbing the interactions along the proposed path, show that Fpg selectively facilitates eversion of oxoG by stabilizing several intermediate states, helping the rapidly sliding enzyme avoid full extrusion of every encountered base for interrogation. Lesion recognition through multiple gating intermediates may be a common theme in DNA repair enzymes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)