Oxidized bases are the major endogenous DNA lesions that can accumulate during aging. Purine and pyrimidine moieties of the respective nucleosides undergo oxidative degradation, resulting in a number of modified bases that can be highly mutagenic when present in DNA. 5OH-Hyd and 5OH-5Me-Hyd residues have been shown to be major oxidation decomposition products of cytosine and thymine, respectively 
. Cells evolved several repair mechanisms to remove oxidized bases from the genome. In the present study, we investigated whether the AP endonucleases involved in the NIR pathway recognize the pyrimidine-derived hydantoins in duplex DNA. The results show for the first time that Nfo, Apn1 and APE1 can incise, in a DNA glycosylase-independent manner, duplex DNA containing both 5OH-Hyd and 5OH-5Me-Hyd residues.
Previous studies of the mechanism of the AP endonuclease-catalyzed nucleotide incision activities were mainly based on the analysis of migration pattern of cleavage fragments in denaturing PAGE 
. Here, to provide insight into the mechanism of NIR activity we analyzed the AP endonuclease-generated cleavage fragments by MALDI-TOF mass spectrometry. The advantage of MALDI-TOF MS analysis is that it permits simultaneous measurements of every DNA products including non-labelled complementary strand, upstream and downstream cleavage fragments. As expected, the results obtained by MALDI-TOF MS perfectly confirmed those obtained by the denaturing PAGE separation technique: all AP endonucleases tested (i
) incise the oligonucleotide duplexes 5′ next to 5OH-Hyd and 5OH-5Me-Hyd residues generating 3′ downstream cleavage fragments still containing 5′-terminal damaged nucleotide; (ii
) degrade 5′ upstream cleavage fragments by their non-specific 3′→5′ exonuclease activity (). Importantly, the MALDI-TOF MS analysis shed new light on the mechanism of nucleotide incision activity on the hydantoins by demonstrating that 5OH-Hyd and 5OH-5Me-Hyd residues in DNA undergo degradation into ureido residues during and/or after incubation with the AP endonucleases. MS data revealed that cleavage of 5OH-Hyd•G and 5OH-5Me-Hyd•A oligonucleotide duplex by all AP endonucleases tested generate DNA fragments containing 5′-terminal ureido residues. Indeed, the denaturing PAGE analysis demonstrated that 3′ downstream cleavage fragments, derived from the treatment of 5OH-Hyd•G, migrate faster than 14-mer size marker fragment but still slower than 13-mer size marker and DNA glycosylase-generated fragments suggesting that 5′-terminal hydantoin residue may undergo partial decomposition (). Formation of ureido residues during AP endonuclease treatment does not depend on reaction condition and incubation time. Furthermore, the co-incubation of 5OH-5Me-Hyd•A duplex with Nfo and APE1 did not increase yield of cleavage fragments containing ureido residues (Supporting Information Figure S1
). Interestingly, the oxidized pyrimidine bases can undergo ring-chain tautomerism at C6-N1 or C5-N1 bond resulting in formation of acyclic linear structures which could be chemically less stable 
. Loss of the base stacking stabilization after duplex incision next to damaged base might change the equilibrium of hydantoins ring-chain tautomerism into the less stable open form. However, MS analysis of P1 nuclease digestion of DNA containing both 5OH-Hyd and/or 5OH-5Me-Hyd residues did not reveal any modification of the hydantoin moiety in the nucleosides 
Previously, we proposed that the NIR activity requires a more tight binding of the AP endonucleases to DNA substrate containing an oxidatively damaged base, as a consequence APE1 has low turnover rate on αdA-containing DNA substrate as compared to AP site DNA 
. This tight mode of binding may enable recognition of oxidized bases in duplex DNA by the AP endonucleases by creating specific interactions of active site amino acid residues with a damaged base. Interestingly, when acting upon 5OH-5Me-Hyd•A, APE1 generates cleavage fragment containing only 5′-ureido nucleotides whereas Nfo and Apn1 produce two fragments containing either 5′-5OH-5Me-Hyd or 5′-ureido nucleotides (). These results indicate that in contrast to Nfo and Apn1, APE1 cannot incise 5OH-5Me-Hyd•A duplex but rather ureido-containing oligonucleotide duplex. Since, NIR-deficient APE1 mutants cannot cleave the hydantoin-containing duplexes (Supporting Information Figure S1A
) and that ureido residue is not present in the non-treated oligonucleotides it is tempting to speculate that under NIR condition APE1 may promote the conversion of 5OH-5Me-Hyd to ureido residue via interactions between its active site amino acids and the damaged pyrimidine. When APE1 binds to DNA it may convert part of 5OH-5Me-Hyd•A to Ureido•A duplex, this would enable APE1 to cut 5′ next to ureido residue generating the observed cleavage fragment with 5′-terminal ureido nucleotide. This is not possible under the BER+Mg2+
condition (in the presence of 5 mM MgCl2
) since under this condition APE1 cannot bind to DNA substrate in the tight manner and catalyze the NIR activity (Supporting Information Figure S2A
). Nfo and Apn1 could also promote the conversion of hydantoin to ureido residue by binding to 5OH-5Me-Hyd•A duplex since they also generate ureido residue after reaction. Interestingly, the co-incubation of 5OH-5Me-Hyd•A duplex with Nfo and APE1 did not increase yield of cleavage fragments containing ureido residues (Supporting Information Figure S1
). Furthermore, ureido residues can be detected by MS after incubation of the 5OH-Hyd•G duplex with all AP endonucleases tested which may suggest conversion of 5OH-Hyd to ureido residue upon enzyme binding to DNA (). Hence, we may speculate that the formation of ureido residues in DNA might be a consequence of both chemical instability of the hydantoins and non-covalent interactions of a damaged base with active site amino acid residues upon AP endonuclease binding. Nevertheless, it should be noted that the degradation of the hydantoins to ureido during or after AP endonuclease-catalyzed cleavage of duplex DNA substrate does not affect removal of the dangling nucleotide residue during reconstitution of the NIR pathway in vitro
, which leads to the restoration of a damage-free duplex oligonucleotide.
In previous studies, we characterized substrate specificities of the bacterial, yeast and human AP endonucleases towards damaged pyrimidines such as DHU, DHT and 5OHC and demonstrated that in vitro
the AP endonucleases are more efficient than the DNA glycosylases/AP lyases 
. In the present work, analysis of kinetic parameters showed that incision of 5OH-Hyd•G by Nfo, Apn1 and APE1 are highly efficient implying that the NIR pathway can efficiently compete with BER in the removal of 5OH-Hyd residues in DNA in vivo
. In contrast, the kinetics parameters of the cleavage of 5OH-5Me-Hyd•A by eukaryotic AP endonucleases Apn1 and APE1 were inefficient as compared to Nfo and DNA glycosylases suggesting that in eukaryotes the majority of 5OH-5Me-Hyd residues would be removed rather in the BER pathway. Interestingly, among all human DNA repair enzymes tested human NTH1 DNA glycosylase has the highest kcat
value for incision of 5OH-5Me-Hyd•A substrate. Therefore, excision of 5OH-5Me-Hyd residues by NTH1 would rather initiate short-patch BER pathway similar to excision of 8oxoG residues by hOGG1 
. Here, based on a new substrate specificity of APE1 we performed a complete in vitro
reconstitution of the human NIR pathway for 5OH-Hyd•G duplex oligonucleotides using purified proteins. Incubation of a 5OH-Hyd•G duplex in the presence of APE1, FEN1, POLβ and LIG1 generated a free of 5OH-Hyd residues, full-length oligonucleotide (). Interestingly, we did not observed futile repair of 5OH-Hyd•G duplex in the presence of DNA ligase activity suggesting that the repair of APE1-generated single-strand breaks should be accomplished through the removal of 5′-dangling nucleotide in the long-patch NIR pathway. Overall, these results demonstrate that 5OH-Hyd residues can be processed in a DNA glycosylase-independent manner via the NIR pathway.
Data obtained with the purified proteins support the physiological relevance of the AP endonuclease-catalyzed nucleotide incision activity on DNA containing pyrimidine-derived hydantoins. To further investigate the role of various DNA repair pathways, we measured the AP endonuclease and DNA glycosylase activities in cell-free extracts from E. coli
and human cells. In E. coli
cell-free extracts we detected mainly DNA glycosylase activities with Nth and Nei being major DNA glycosylases responsible for incision of 5OH-Hyd•G and 5OH-5Me-Hyd•A duplexes and little NIR activity (). Although, the Nfo-catalyzed NIR activity towards 5OH-Hyd•G and 5OH-5Me-Hyd•A can be strongly induced by paraquat up to the level similar to those observed for DNA glycosylases (Supporting Information Figure S4
). Interestingly, it was shown that E. coli nth nei
mutants are hypersensitive to the lethal effects of ionizing radiation 
and hydrogen peroxide 
, implying potential role of pyrimidine-derived hydantoins as lethal oxidative lesions in DNA. In the case of human cell-free extracts, depending on the reaction conditions either NIR+Zn2+
and/or BER+EDTA activities were detected (). Using small RNA silencing we demonstrated that the alternative DNA glycosylase-independent repair of 5OH-Hyd and 5OH-5Me-Hyd residues in duplex DNA depends upon APE1 thus substantiating the biological role of APE1-catalyzed NIR pathway in human cells (). Recently, it has been demonstrated that Nei and NEIL1 mediated excision of 5OH-5Me-Hyd can result in an unproductive DNA–protein covalent (DPC) complex which hides the lesion from repair and represents more complex bulky lesion 
. This observation further substantiates the biological role of NIR as an alternative pathway which avoids the generation of genotoxic intermediates during repair of the hydantoin DNA lesions.
Under BER (BER+EDTA and BER+Mg2+
) conditions, three human DNA glycosylases can excise 5OH-Hyd and 5OH-5Me-Hyd residues hence contributing to the redundancy in DNA repair pathways that may back-up each other and/or act preferably depending on chromatin context, DNA damage signalling pathway and various cellular regulation mechanisms. Study of the BER activities in HeLa cell extracts demonstrated that NTH1 is a major detectable DNA glycosylase activity towards 5OH-Hyd and 5OH-5Me-Hyd residues in DNA (). Surprisingly, we were not able to detect NEIL1 and NEIL2 activities using our hydantoin-DNA substrates possibly due to a strong 3′-repair diesterase activity present in human cell-free extracts. Human FA cells appear to be a highly valuable model to study cellular response to endogenous oxidative DNA damage. Ambient oxygen induces chromosomal instability in FA cells suggesting impaired cellular defence against oxidative DNA damage, furthermore we have recently shown that FA cells have reduced amounts of NEIL1 
. Interestingly, here we demonstrated that FA cell-free extracts have slightly reduced BER incision activity towards 5OH-Hyd•G duplex oligonucleotide implying that NEIL1 may serve as a back-up DNA glycosylase to repair pyrimidine-derived hydantoins (Supporting Information Figure S5
). Human NTH1 protein has been shown to be able to initiate BER in nucleosome protected DNA 
, while NEIL1 and NEIL2 proteins excise oxidative base lesions in single-stranded and bubble DNA structures, suggesting their functions are coupled to DNA replication and/or transcription processes 
. 5OH-Hyd and 5OH-5Me-Hyd residues are major oxidative pyrimidine lesions that accumulate in ancient DNA and may also accumulate during long chronic exposure to oxidizing agents 
. Therefore, it is tempting to speculate that pyrimidine-derived hydantoins in non-transcribed heterochromatin DNA regions are main targets to NTH1 and APE1 but not to NEIL1, suggesting biological function of the NTH1-catalyzed BER and the APE1-NIR in the global genome repair pathway for pyrimidine-derived hydantoins elsewhere in genome.