Construction and characterization of three- and four-stranded DNA structures containing a single ICL

To further characterize DNA substrate specificities of the Nei, NEIL1 and NEIL3 DNA glycosylases, we constructed and characterized long, three- and four-stranded DNA structures containing a single psoralen-derived ICL. These crosslinked DNA structures mimic the products of DNA translesion synthesis bypass of an ICL site in duplex DNA. The migration pattern of all DNA structures was first analyzed by non-denaturing PAGE (Fig. 1). Oligonucleotide sequences used in this work are shown in Supplementary Table ^S2. To start the construction, the 5′-[³²P]-labelled single-stranded 21 and 47 mer DNA oligonucleotides, referred to as D21 and C47, respectively (containing a single 5′-TpA site at position 9 and 24, respectively), were annealed to the partially complementary 21, 47 and 101 mer oligonucleotides, referred as C21/D21, D47 and D101, respectively (containing a single 5′-TpA site at position 12/9, 23 and 49, respectively), to obtain non-covalent D21*∙C21, D21*∙C47/C47*∙D21 and C47*∙D101 duplex oligonucleotides (“*”denotes the 5′-[³²P]-labelled oligonucleotide), respectively (Fig. 1, panels E, I and N). The oligonucleotide duplexes were exposed to either HMT+UVA or 8-MOP+UVA treatments and the resulting 5′-[³²P]-labelled crosslinked DNA duplexes XL21-21, XL47-21 and XL101-47 were purified by denaturing PAGE (Fig. 1, panels F, J and O). Next, the purified XL47-21 and XL101-47 oligonucleotides containing a single ICL were hybridized to complementary D47 and C101 DNA oligonucleotides to obtain 5′-[³²P]-labelled three-stranded DNA structures, XL47∙47-21 and XL101∙101-47 (Fig. 1, panels G, K, O and P). Finally, the three-stranded XL47∙47-21 oligonucleotides were hybridized to C21, to obtain the 5′-[³²P]-labelled four-stranded DNA XL47∙47-21∙21 oligonucleotides (Fig. 1, panels H and L). Analysis of the migration pattern showed that the single-stranded oligonucleotides migrate faster than the duplex structures while the crosslinked duplexes migrate slightly slower than their non-crosslinked counterparts. The three-stranded crosslinked DNA structures XL47∙47-21 and XL101∙101-47 (lanes 7, 8, 12 and 17) migrate much slower than the respective crosslinked duplexes XL47-21 and XL101-47 (lanes 6, 11 and 16). And finally, the four-stranded crosslinked DNA structures XL47∙47-21∙21 (lanes 9 and 13) migrate even more slowly than their respective three-stranded crosslinked DNA structures XL47∙47-21 (lanes 7, 8 and 12). We observed smearing of DNA bands on gel electrophoresis corresponding to the D, I, M, N, O, P and Q substrates (lanes 4 and 14−17) which may be due to secondary structures formed by C47 oligonucleotide (lane 4). Taken together, the analysis of the migration patterns of crosslinked DNA duplexes on a non-denaturing gel suggest the formation of stable three- and four-stranded DNA structures.

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Figure 1

Non denaturing PAGE analysis of the DNA structures containing single ICL used in DNA glycosylase activity assays. 5′-[³²P]-labelled oligonucleotide strands are denoted by “*”, the presence of a crosslink is denoted by “-” and normal oligonucleotide base complementation is indicated by “∙”.

NEIL3 excises an unhooked ICL within a three-stranded DNA structure

Recently, it has been shown that Xenopus NEIL3 is able to excise thymine and adenine nucleotides crosslinked to a single-stranded oligonucleotide, suggesting that the NEIL3 homologs can remove psoralen-generated ICLs when present in DNA²⁸. To examine whether human NEIL3 excises a psoralen-induced ICL in three-stranded DNA structures, we utilised the 5′-[³²P]-labelled XL47∙47-21* and XL47∙47*−21 oligonucleotides (“*”denotes the 5′-[³²P]-labelled strand) containing a 21 mer oligonucleotide crosslinked by HMT+UVA exposure to a 47 mer duplex (see Fig. 1, panels G2 and K). To identify DNA glycosylase cleavage products we prepared oligonucleotide size markers containing: (i) a single psoralen-derived thymine monoadduct (MA), for this, an HMT-crosslinked XL21*−21 and XL47*−21 duplexes were incubated under hot-alkali conditions²⁹; (ii) 3′-terminal phosphate (P); (iii) 3′-terminal hydroxyl group (OH); and (iii) 3′-terminal α,β-unsaturated phosphoaldehyde (PA) (for details see Materials and Methods). Structure of DNA modifications used in this work are shown in Supplementary Table ^S1. As expected, incubation of the 5′-[³²P]-labelled XL47∙47-21* (panel G2) with NEIL1 generated a 21 mer cleavage product (Fig. 2A, lane 5) that migrated slightly more slowly than the 21 mer with MA (21-MA) and regular 21 mer D21 oligonucleotide (lanes 6 and 12). This result suggests that NEIL1: (i) remove a long crosslinked 21 mer oligomer from the XL47∙47-21* structure via excision of the crosslinked thymine base in the 47 mer duplex oligonucleotide; and (ii). generate a 5′-[³²P]-labelled 21 mer ssDNA fragment containing a deoxythymidine monophosphate (dTMP) nucleotide crosslinked to an extrahelical thymine residue (Supplementary Table ^S1) and non-labelled nicked 47 mer duplex oligonucleotide. However, no cleavage of the 21 mer excision product was observed after incubation with NEIL1 suggesting that these DNA glycosylases recognize crosslinked thymine only within 47 mer duplex DNA.

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Figure 2

Action of Nei-like DNA glycosylases upon three-stranded DNA structure containing single HMT-derived ICL. (A) Denaturing PAGE analysis of the reaction products. 10nM 5′-[³²P]-labelled XL47∙47-21* was incubated for 1 hr at 37°C either with the 500nM NEIL3^Cat, NEIL3^Trun, NEIL3^FL or with 50nM NEIL1 proteins. Lanes 1–7, no piperidine treatment; lane 1, control non-treated XL47∙47-21*; lanes 2–4, as 1 but with NEIL3s; lane 5, NEIL1; lane 6, 21 mer duplex containing MA; lane 7, as 6 but Nei; lanes 8–11, as 1–4 but treated with light piperidine; lane 12, 21 mer D21; lanes 13–15, 21 mer U∙A duplex treated with UDG/hot piperidine, UDG/Nfo and UDG/light piperidine, respectively. Substrate and cleavage products sizes are indicated to the right of the gel. “X”denotes substrate, “21-MA” denotes 21 mer fragment containing psoralen-derived MA, “21-mer” denotes size marker, “8^PA”, “8^OH” and “8^P” denote 8 mer fragments containing 3′-terminal PA, OH and P, respectively. For details see Materials and Methods. (B) Graphical representation of NEIL3s protein concentration dependent activities upon three-stranded DNA structure.

Incubation of the 5′-[³²P]-labelled XL47∙47-21* (Fig. 1, panel G2) with NEIL3^Cat and NEIL3^Trun without piperidine treatment resulted in the appearance of 21 mer excision fragments with a smeared migration pattern below suggesting the unhooking of the ICL in XL47-21 and formation of an abasic site in the released 21 mer D21* oligonucleotide (Fig. 2A, lanes 2 and 3). Incubation with NEIL3^FL generated cleavage products but with very low efficiency compared to the truncated proteins (lane 4). Here we used piperidine treatment to cleave AP sites generated by NEIL3. It should be noted that piperidine is a highly efficient β-catalyst that cleaves AP sites in DNA either via β-elimination at 37°C or via β-δ elimination reaction at 90°C^30-32. Light piperidine treatment of the NEIL3 reaction products resulted in the formation of an 8 mer cleavage product (lanes 9–11) that migrated similar to the 8^PA mer size marker (lane 15) but much more slowly than the 8^OH size marker (lane 14) and 8^P mer product generated by Nei acting upon the 21 mer duplex containing an HMT-derived MA (lane 7). However, piperidine treatment did not result in the disappearance of free 21 mer D21* oligonucleotide suggesting that it still contains psoralen-derived adduct (lanes 9–11). These results suggest that the human NEIL3 proteins excise a long 21 mer single-stranded oligomer crosslinked to duplex DNA in XL47∙47-21* by hydrolysing the glycosidic bond between the crosslinked thymine and deoxyribose of either the 5′-[³²P]-labelled D21 oligonucleotide or non-labelled 47 mer C47∙D47 duplex. It should be noted that incubation of non-treated XL47∙47-21* in light piperidine resulted in partial decross-linking with the appearance of 21-MA and 21 mer fragments (lane 8). The HMT-induced ICLs can be cleaved and converted to mono-adducts by alkaline treatment and the efficiency of decross-linking depends on temperature and nature of the alkali used²⁹.

To assess relative efficiencies of the NEIL3 proteins on ICLs, we examined enzyme concentration dependent cleavage of XL47∙47-21*. As shown in Fig. 2B, NEIL3^Cat was the most efficient ICL-excising DNA glycosylase, when compared to the other NEIL3 proteins, cleaving 38% of the DNA at the highest enzyme concentration. Whereas, NEIL3^Trun cleaved 10% and NEIL3^FL exhibited very weak activity, cleaving only 2% of DNA substrate under the same reaction conditions. Similar results were obtained when incubating the NEIL3 proteins with the 8-MOP-derived three-stranded XL47∙47-21* (Fig. 1, panel G1 and Supplementary Fig. ^S3) suggesting that NEIL3 can recognize ICLs generated by different psoralens. As expected, no cleavage products were observed when the 5′-[³²P]-labelled XL47∙47-21* was incubated with increasing amounts of the catalytically deficient NEIL3^Cat-K81A mutant with or without piperidine treatment (Supplementary Fig. ^S4). Thus, NEIL3 can target HMT and 8-MOP crosslinked thymine either in the unhooked single-stranded portion or in duplex DNA of the three-stranded DNA structure and generate a free ssDNA fragment and duplex DNA, each containing either an abasic site or dTMP crosslinked to an extrahelical thymine residue, respectively.

To further substantiate the mechanisms of action of Neil-like DNA glycosylases, we used the three-stranded XL47∙47*−21 oligonucleotide substrate in which the 47 mer C47 oligonucleotide was 5′-[³²P]-labelled (Fig. 1, panel K). Incubation of the 5′-[³²P]-labelled XL47∙47*−21 with NEIL3s resulted in the formation of 47 mer and 23^PA mer cleavage products, whereas incubation with Nei generated a 23^P mer product only (Supplementary Fig. ^S5). Again these results suggest that in contrast to Nei, human NEIL3 can recognize crosslinked thymine residues in both the duplex and single-stranded oligonucleotide portions of the three-stranded DNA structure. Schematic representation of the mechanism of action of Nei-like DNA glycosylases upon three-stranded DNA structure is shown in Fig. 3A.

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Figure 3

Schematic representation of the mechanisms of action of Nei-like DNA glycosylases on ICLs. (A) Mechanisms of action of Nei-like DNA glycosylases on three-stranded DNA structure containing single psoralen-derived ICL. For details see text. (B) Mechanisms of action of Nei-like DNA glycosylases on four-stranded DNA structure containing single psoralen-derived ICL. (C) Skeletal formula of 21 mer fragment containing single Thymidine nucleotide crosslinked to free Thymine base by HMT (DNA(T)-HMT-T), an excision product generated by Nei-like DNA glycosylases catalyzed repair of ICL in three-stranded DNA structure. (D) Skeletal formula of Thymine-HMT-Thymine cross-link (T-HMT-T), an excision product generated by Nei-like DNA glycosylases catalyzed repair of ICL in three- and four-stranded DNA structure.

Next we examined whether the length of the crosslinked DNA fragment is critical for recognition of an ICL by the Nei-like DNA glycosylases. For this we constructed the three-stranded 5′-[³²P]-labelled XL101∙101-47* oligonucleotide (“*”denotes the 5′-[³²P]-labelled strand) containing a 47 mer C47 oligonucleotide crosslinked to a 101 mer C101∙D101 duplex (see Fig. 1, panel P). As expected, incubation of XL101∙101-47* with Nei, NEIL1 generated a ~47 mer cleavage product that migrated more slowly than the non-modified 47 mer C47 oligonucleotide (Supplementary Fig. ^S6). Whereas, incubation of the 5′-[³²P]-labelled XL101∙101-47* with NEIL3^Cat, NEIL3^Trun and NEIL3^FL followed by piperidine treatment resulted in the appearance of 47 mer and 23 mer cleavage products, the latter migrated more slowly than the 23 mer product generated by Nei when acting upon the MA containing 47 mer, C47*∙D47 duplex (Supplementary Fig. ^S6). These results are in agreement with the above data and suggests that Nei-like DNA glycosylases can remove a long crosslinked 47 mer oligomer from the XL101∙101-47* structure via excision of a crosslinked thymine either in the 101 mer duplex or in the 47 mer single-stranded oligomer.

Nei-like DNA glycosylases cleave ICLs in a four-stranded DNA structure

It has previously been shown that the specialized TLS DNA polymerases, such as Polκ, Polη and Polν^33–35 can synthesize across an ICL in duplex DNA with subsequent primer extension by the REV1/Polζ complex^36–39. Additionally, the DNA translocase FANCM can promote replication fork traverse across ICLs in DNA, which enables the stalled replication fork to restart past the ICL and continue DNA synthesis without lesion repair²². Based on these observations, we may hypothesize further by suggesting that DNA replication can directly bypass an ICL, without the involvement of incision steps to unhook the ICL, either on one DNA strand to generate an uninterrupted three-stranded DNA structure, or on both DNA strands to generate a four-stranded DNA structure still containing the unrepaired ICL. Here, we examined whether a four-stranded DNA structure composed of two duplex DNA molecules crosslinked to each other by HMT or 8-MOP are substrates for Nei family DNA glycosylases. For this, we constructed the four-stranded 5′-[³²P]-labelled XL47∙47-21*∙21 and XL47∙47*−21∙21 oligonucleotides containing the 21 mer D21∙C21 oligonucleotide duplex crosslinked to the 47 mer C47∙D47 duplex (see Fig. 1, panels H and L). Incubation of XL47∙47-21*∙21, containing an HMT-derived ICL, with E. coli Nei generated a major 8 mer cleavage product (Fig. 4, lane 5) which migrated similar to the 8^P mer size marker containing a 3′-phosphate (lane 13) and the 8^P mer cleavage product generated by Nei when acting upon the 21 mer duplex containing MA (lane 7). Whereas, incubation of XL47∙47-21*∙21 with human NEIL3^Cat and piperidine generated 8 mer and 21 mer cleavage products (lane 9), the former migrated similar to the 8^PA mer size marker (lane 15) and the latter migrated similar to the 21 mer fragment containing MA (lane 6). Interestingly, the NEIL3-released 21 mer excision product did not disappear after piperidine treatment (lanes 9–10), but migrated similar to 21 mer fragment containing MA (lane 6) but more slowly than the 21 mer size marker (lane 12). As expected, no cleavage products were observed when the four-stranded 5′-[³²P]-labelled XL47∙47-21*∙21 was incubated with increasing amounts of the catalytically deficient NEIL3^Cat-K81A mutant with or without piperidine treatment (Supplementary Fig. ^S7). Altogether, these results suggest that the Nei-like DNA glycosylases are able to repair an ICL within a four-stranded DNA structure by excising crosslinked thymine, either in a short, labelled 21 mer D21*∙C21 duplex, or in a long non-labelled 47 mer C47∙D47 duplex.

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Figure 4

Action of Nei-like DNA glycosylases upon four-stranded DNA structure containing single HMT-derived ICL. Denaturing PAGE analysis of the reaction products. 10nM 5′-[³²P]-labelled XL47∙47-21*∙21 was incubated for 1 hr at 37°C either with the 500nM NEIL3^Cat, NEIL3^Trun, NEIL3^FL or with 20nM Nei proteins. Lanes 1–7, no piperidine treatment; lane 1, control non-treated XL47∙47-21*∙21; lanes 2–4, as 1 but with NEIL3s; lane 5, as 1 but Nei, lane 6, 21 mer duplex containing MA; lane 7, as 6 but Nei; lanes 8–11, as 1–4 but treated with light piperidine; lane 12, 21 mer D21; lanes 13–15, 21 mer U∙A duplex treated with UDG/hot piperidine, UDG/Nfo and UDG/light piperidine, respectively. Substrate and cleavage products sizes are indicated to the right of the gel. “X” denotes substrate, “21-MA” denotes 21 mer fragment containing psoralen-derived MA, “21-mer” denotes size marker, “8^PA”, “8^OH” and “8^P” denote 8 mer fragments containing 3′-terminal PA, OH and P, respectively. For details see Materials and Methods.

Similar results were obtained when incubating the Nei-like DNA glycosylases with the HMT-derived four-stranded XL47∙47*−21∙21 substrate in which the 47 mer C47 oligonucleotide was 5′-[³²P]-labelled (Fig. 1, panel L and Supplementary Fig. ^S8) and also with the 8-MOP-derived XL47∙47-21*∙21 (where either 21 mer C21 or D47 mer is 5′-[³²P]-labelled) (Supplementary Fig. ^S9). Thus, action of Nei and NEIL1 on four-stranded DNA generates two free duplex molecules each containing a single-strand break and two thymine bases crosslinked to each other as end products of the reaction. Whereas, action of NEIL3 on four-stranded DNA generates two free duplex molecules, one containing an abasic site and the other containing a dTMP crosslinked to an extrahelical thymine base. Schematic representation of the mechanism of action of Nei-like DNA glycosylases upon four-stranded DNA structure is shown in Fig. 3B.

Expression and purification of DNA glycosylases

The purified BER proteins including human NEIL1, E. coli uracil-DNA glycosylase (UDG), endonuclease IV (Nfo) and Nei were from laboratory stock. The activities of various DNA repair proteins were tested using their principal substrates and were checked just prior to use. The bacterial expression vector phNEIL1 was generously provided by Drs Hiroshi Ide (Hiroshima University, Japan)⁴². The full length native NEIL1 was purified as described previously⁴². The expression vector pET13a-Nei for the E. coli Nei protein was generously provided by Dmitry Zharkov (ICBFM, Novosibirsk, Russia)⁴³. The E. coli Nei protein was purified as described previously⁴⁴.

Purification of human NEIL3 proteins. As the N-terminal methionine residue adjacent to catalytic valine of NEIL3 is poorly removed during protein maturation in host bacterial cells, Wallace and colleagues developed a protocol in which they co-expressed a modified E. coli methionine aminopeptidase (EcoMap, Y168A) with mammalian NEIL3 proteins to increase the amount of active DNA glycosylase in the preparation⁴⁵. Expression and purification of recombinant hNEIL3 proteins was achieved by the molecular cloning of hNEIL3^Cat, ^Trun and ^FL cDNA sequences into the pETDuet2-EcoMap-ORF6- bicistronic expression vector (a generous gift from S.S. Wallace to J.L. Parsons). Full details of the cloning, expression and purification will be reported elsewhere. Vectors were transformed into Rosetta 2 (DE3) expression hosts for recombinant protein expression and subsequent FPLC purification following similar methods to those described by Liu and colleagues⁴⁵.

To prepare the hNEIL3^Cat-K81A-His expression vector, the pETDuet2-EcoMap-ORF6-hNEIL3^Cat expression vector was subjected to site directed mutagenesis using 1.75 units of Pfu Ultra DNA polymerase (Agilent technologies). PCR primers were used, to mutate the codon encoding for a lysine residue at amino acid position 81 to an alanine, to remove glycosylase and AP lyase activity as described by Krokeide and colleagues¹⁵. Products were digested with 10 units of DpnI restriction endonuclease and transformed into Nova XG cloning hosts by electroporation and confirmed by Sanger sequencing as previously described. Expression and purification of hNEIL3^Cat-K81A was carried out following the same methods used for purification of hNEIL3^Cat.

Preparation of psoralen ICLs

30 pmol of 5ʹ-[³²P]-labelled D21 or C47 oligonucleotide were mixed with 120 pmol of non-labeled D21 or C47 oligonucleotide, respectively. 130 pmol of total D21 or C47 oligonucleotide was mixed with 143 pmol of C47 and D47 or D101 respectively and annealed, generating 47∙21*, 47*∙21 or 101∙47* oligonucleotide duplexes. 117 pmol of total oligonucleotide from the annealing reactions was incubated with 0.5µM HMT or 8-MOP for 15min in the dark at room temperature in dH₂O at a final volume of 100µl. Samples were then irradiated at 365nm and 240kJ/m2 for 30min (HMT) or 60min (8-MOP) on ice. Crosslinked oligonucleotides were then separated by 20% denaturing-PAGE and DNA bands containing psoralen crosslinked oligonucleotides were excised. It should be noted that photoreaction of asymmetric psoralens with the DNA duplex yields two orientational isomers of the ICL that migrate in denaturing PAGE as two bands^29,46. Crosslinked oligonucleotides were eluted from gel strips in dH₂O and precipitated in 2% LiClO₄/acetone. Eluted crosslinked oligonucleotides were reconstituted in dH₂O for use in further annealing reactions, oligonucleotide substrate analysis and DNA glycosylase activity assays. Preparation of all psoralen adducted oligonucleotides were conducted in the same manner.

To prepare three- and four-stranded crosslinked DNA oligonucleotides containing psoralen-derived ICLs, the XL47-21 and XL101-47 duplexes were annealed with complementary 21, 47 and 101 mer oligonucleotides, respectively.

DNA repair assay

The standard reaction mixture (20µL) for ICL-specific DNA glycosylase activity contained 10nM of 5´-[³²P]-labelled DNA substrate, incubated with reaction specific concentrations of enzyme, diluted in RDWB buffer (25mM HEPES-KOH pH 7.6, 100mM KCl, 1mM EDTA, 1mM DTT and 17% glycerol) and DNA glycosylase reaction buffer (25mM HEPES-KOH pH 7.6, 100mM KCl, 1mM EDTA, 1mM DTT and 100µg/ml BSA) for 60min at 37°C. Reactions were stopped by the addition of 0.2% SDS and 4mM EDTA and incubated if necessary for a further 30min at 37°C in the presence of 10% piperidine, to cleave AP sites by β-elimination. The reaction was then stopped with the addition of 20mM EDTA and neutralised with 40mM HCl. The samples were desalted by passing through house-made spin-down columns filled with Sephadex G25 (GE Healthcare) equilibrated in 7.5M urea. Desalted reaction products were separated by electrophoresis in denaturing 20% (w/v) polyacrylamide gel (7.5M urea, 0.5×TBE), gels were exposed to a Fuji FLA-9500 Phosphor Screen and analyzed using Image Gauge V3.12 software.

Preparation of DNA size markers containing 3′-terminal modifications

The DNA fragments containing 3′-terminal phopshate (P), hydroxyl (OH) and α,β-unsaturated phosphoaldehyde (PA) groups were prepared by the cleavage of duplex oligonucleotide containing a single AP site with hot piperidine, E. coli Nfo and light piperidine, respectively. For this 5ʹ-[³²P]-labelled D21-U and C47-U oligonucleotides containing a single Uracil residue at position 9 and 24, respectively, were hybridized with complementary regular C21 and D47 oligonucleotides to obtain 21 and 47 mer duplexes with single U∙A base pair. The U∙A duplexes were incubated in the presence of 20nM E. coli UDG in DNA glycosylase reaction buffer for 30min at 37°C to obtain AP sites. Then 21 and 47 mer duplexes with an abasic site were incubated for 30min at 37°C in the presence of 10% piperidine to cleave AP sites via β-limination and produce 5ʹ-[³²P]-labelled 8 and 23 mer cleavage fragments containing 3′-terminal PA group. Treatment by hot piperidine (30min at 90°C in the presence of 10% piperidine) incised AP sites in DNA via β,δ-elimination and produced 5ʹ-[³²P]-labelled 8 and 23 mer cleavage fragments containing 3′-terminal P group. Hydrolytic cleavage of AP sites in DNA by bacterial AP endonuclease Nfo produced 5ʹ-[³²P]-labelled 8 and 23 mer cleavage fragments containing 3′-terminal OH group. Structures of DNA fragments with 3′-terminal modifications are shown in Supplementary Table ^S1.

In addition, we incubated 5ʹ-[³²P]-labelled 21 mer D21*∙C21 and 47 mer C47*∙D47 duplexes containing a single MAp adduct with Nei to generate 8 and 23 mer cleavage fragments containing 3′-terminal P group.

Equipment and settings

The acquisition of images of denaturing PAGE and non-denaturing PAGE gels was facilitated by exposure to a Storage Phosphor Screen BAS (GE Healthcare Life Sciences) overnight. Phosphor Screens were then imaged using the Typhoon^TM FLA 9500 Laser Scanner (GE Healthcare Life Sciences) with an pixel resolution of 100µM. Retrieved images in TIFF format were then processed in Adobe photoshop CS6 software, to adjust brightness and contrast settings for appropriate visualisation of 5′-³²P labelled DNA. Consistent adjustment of brightness and contrast was used across the entirety of the gel image. Quantification of DNA cleavage was facilitated by use of the FUJIFILM Science Lab Image Gauge Ver. 4.0 software (Fujifilm), with quantification adjusted by removal of standardised background values on each gel image. GraphPad Prism 7 software was used for generation of graphical representations of percentage cleavage quantifications. Schema were generated with use of Adobe photoshop CS6. SDS-PAGE image showing final purification products was imaged at 700nm wavelength using the Licor Odyssey Infared imaging system.

We are grateful to Susan S. Wallace (University of Vermont, USA), Hiroshi Ide (Hiroshima University, Japan), Dmitry O. Zharkov (ICBFM, Novosibirsk, Russia) and Nicolas E. Geacintov (New York University, USA) for the DNA glycosylase expression constructs and modified oligonucleotide, respectively. This work was supported by grants from la Ligue Nationale Française Contre le Cancer « Equipe LNCC 2016 » and Electricité de France RB 2016-17 (to M.S.), North West Cancer Research (CR972, CR1016, CR1074 and CR1145) and by the Medical Research Council via a New Investigator Research Grant MR/M000354/1 (to J.L.P.) and Nazarbayev University ORAU and the Ministry of Education and Science of the Republic of Kazakhstan programs 0115RK02473 and 0115RK03029 (to B.T.M). P.R.M. and M.S.A. were the recipients of a ‘Pathway to Excellence′ PhD studentship from the University of Salford, U.K. and a PhD studentship awarded by the Ministry of Higher Education to the Biotechnology Research Centre (BTRC), Tripoli, Libya, respectively. C.Z. was supported by fellowship MESR, France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.