International Journal of Molecular Sciences

Article Complementary Functions of Plant AP Endonucleases and AP during DNA Repair of Abasic Sites Arising from C:G Base Pairs

Marina Jordano-Raya 1,2,3, Cristina Beltrán-Melero 1,2,3 , M. Dolores Moreno-Recio 1,2,3, M. Isabel Martínez-Macías 1,2,3, Rafael R. Ariza 1,2,3 , Teresa Roldán-Arjona 1,2,3 and Dolores Córdoba-Cañero 1,2,3,*

1 Department of , University of Córdoba, 14071 Córdoba, Spain; [email protected] (M.J.-R.); [email protected] (C.B.-M.); [email protected] (M.D.M.-R.); [email protected] (M.I.M.-M.); [email protected] (R.R.A.); [email protected] (T.R.-A.) 2 Maimónides Biomedical Research Institute of Córdoba (IMIBIC), 14004 Córdoba, Spain 3 Reina Sofía University Hospital, University of Córdoba, 14004 Córdoba, Spain * Correspondence: [email protected]

Abstract: Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from sponta- neous base loss and excision of damaged bases. They may be processed either by AP endonucleases   or AP lyases, but the relative roles of these two classes of are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence sur- Citation: Jordano-Raya, M.; Beltrán-Melero, C.; rounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the Moreno-Recio, M.D.; activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an Martínez-Macías, M.I.; Ariza, R.R.; abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common Roldán-Arjona, T.; intermediates during the repair of deaminated , whereas AP sites opposite C frequently Córdoba-Cañero, D. Complementary arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited Functions of Plant AP Endonucleases a higher efficiency on AP sites opposite G. In contrast, the main plant AP (FPG) showed a and AP Lyases during DNA Repair of greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as Abasic Sites Arising from C:G Base the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and Pairs. Int. J. Mol. Sci. 2021, 22, 8763. AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing https://doi.org/10.3390/ijms the potential mutagenic consequences of C and G oxidation, respectively. 22168763

Keywords: abasic sites; AP endonucleases; AP lyases; ; Arabidopsis Academic Editor: Jean Molinier

Received: 27 July 2021 Accepted: 11 August 2021 Published: 16 August 2021 1. Introduction Abasic (apurinic/apyrimidinic, AP) sites are inescapable DNA lesions arising by Publisher’s Note: MDPI stays neutral spontaneous hydrolysis of the N-glycosylic bond between intact and deoxyri- with regard to jurisdictional claims in bose [1]. Spontaneous base release is additionally facilitated by some alterations induced by published maps and institutional affil- genotoxic compounds. For example, methylation of guanine (N7-methylguanine, N7-meG) iations. results in weakening of the N-glycosylic bond and a marked increase in base loss [2–4]. AP sites are also enzymatically generated as intermediates during the Base Excision Repair (BER) pathway, which is initiated by DNA glycosylases that catalyse the excision of modi- fied bases from DNA [5–8]. It has been estimated that mammalian cells have steady-state Copyright: © 2021 by the authors. levels of 50,000–200,000 AP sites per under physiological conditions [9]. AP sites Licensee MDPI, Basel, Switzerland. exist as an equilibrium mixture of hemiacetals of the closed furanose form, but approxi- This article is an open access article mately 1% is present as the ring-opened aldehyde species which is prone to spontaneous distributed under the terms and hydrolysis and may generate single-strand breaks (SSBs) [10,11]. Unrepaired AP sites are conditions of the Creative Commons cytotoxic since they block DNA replication and transcription. DNA replication blockage Attribution (CC BY) license (https:// may be avoided through translesion DNA synthesis across the AP site, which usually creativecommons.org/licenses/by/ results in [12,13]. 4.0/).

Int. J. Mol. Sci. 2021, 22, 8763. https://doi.org/10.3390/ijms22168763 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 8763 2 of 15

AP sites are generally repaired through the BER pathway [6,14,15], although other DNA repair routes, such as Nucleotide Excision Repair (NER) may contribute as backup mechanisms [16]. The repair of an AP site through BER requires the removal of the deoxyri- bose phosphate moiety from DNA to allow the insertion of an intact deoxyribonucleotide. Such removal may be initiated by two distinct classes of enzymes: AP endonucleases and 0 AP lyases [17]. AP endonucleases cleave the phosphodiester bond at the 5 side of the AP 0 0 site, generating a strand break with a free 3 -OH terminus and a blocking 5 -deoxyribose 0 0 phosphate (5 -dRP) end. In contrast, AP lyases perform the incision at the 3 side of the AP site by cleaving the sugar moiety through a β-elimination mechanism that generates 0 0 0 a blocking 3 -phosphor-α,β-unsaturated aldehyde (3 -PUA) and a free 5 -P terminus. A 0 0 subset of AP lyases perform a β,δ-elimination; thus, generating a blocking 3 -P end. The 5 - 0 and 3 -blocked ends generated by the incision activity of AP endonucleases and AP lyases, respectively, are removed by downstream enzymes before gap filling and ligation achieve a full repair [7,8]. AP lyase activity is usually found in the so-called bifunctional DNA glycosylases, which are able to incise the AP site generated by their own N-glycosylase activity. The remaining DNA glycosylases lack such capability and are called monofunctional [18]. The biological relevance of the AP lyase activity of bifunctional DNA glycosylases (also termed DNA glycosylases/AP lyases) is not well understood. In particular, it has been a long-standing question whether such enzymes are able to process in vivo AP sites not generated by their own DNA glycosylase activity [19]. Based on evidence chiefly obtained in mammalian cells, it has been generally accepted that, in vivo, the vast majority of AP sites, either from spontaneous or enzymatic origin, are repaired by AP endonucleases [20]. However, studies in non-mammalian systems point to a physiological role for AP lyases in removing AP sites arising independently of N-glycosylase activity. In S. pombe, for example, most abasic sites are incised by the AP lyase activity of the bifunctional DNA 0 glycosylase Nthp1, generating nicks with 3 -PUA ends that are converted to 30-OH by the activity of Apn2, the major AP endonuclease in fission yeast [21,22]. Results obtained in S. cerevisiae suggest an analogous scenario, in which the AP lyase activity of Nthp1 homologs Ntg1 and Ntg2 acts upstream of AP endonucleases Apn1 and Apn2 during repair of AP sites [23]. Thus, AP endonucleases in yeast may predominantly function in the removal of 30-blocks generated by AP lyases. Interestingly, it has been recently reported that the phosphodiesterase activity of human APE1 plays a relevant role in processing 30-PUA ends generated by the lyase activity of NTHL1 in nucleosomal, but not in naked, DNA [24]. Additionally, it has been shown that the bifunctional DNA glycosylase NEIL2, which is upregulated in the breast cancer cell line Hs578T, outcompetes APE1 at AP sites and sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations [25]. In plants, we recently reported that FPG, the major AP lyase of Arabidopsis thaliana, has a relevant biological role in the repair of AP sites generated by the spontaneous release of N7-meG [26]. Such AP sites are very poor substrates for ARP, the major AP endonuclease in Arabidopsis, but are efficiently incised by the AP lyase activity of FPG, a β, δ-elimination cat- alyst. The blocking 30-P ends generated by FPG are processed by the DNA 30-phosphatase ZDP, allowing to complete repair in an AP endonuclease-independent pathway [26]. In Arabidopsis, both ARP and FPG incise enzymatically generated AP sites, but the factors implicated in the choice between endonuclease- or lyase-initiated repair remain unknown. For some BER enzymes targeting the same lesion, for example, different DNA glycosylases, two important specificity factors are the flanking sequence and the identity of the opposite base on the complementary strand. Thus, in Arabidopsis, both UNG and MBD4L DNA glycosylases excise uracil, which commonly arises from spontaneous C deamination [1]. However, whereas UNG displays flexibility for the opposite base and the flanking sequence [27], MBD4L only excises U when opposite G and, additionally, shows a strong preference for a DNA sequence context (50-CG-30) with a high probability of methylation [28]. We proposed that MBD4L has evolved to specifically counteract C and 5-meC deamination at CG sequences, where most plant DNA methylation is found [28]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 16

of cytosine methylation [28]. We proposed that MBD4L has evolved to specifically coun- teract C and 5-meC deamination at CG sequences, where most plant DNA methylation is Int. J. Mol. Sci. 2021, 22, 8763 3 of 15 found [28]. Additionally, the Arabidopsis 5-methylcytosine (5-meC) DNA glycosylase ROS1 efficiently excises T (= 5-methyluracil, 5-meU) but only at T:G mismatches, and also displays a strong preference for a CG sequence context [29,30]. Therefore, the specificity Additionally,of some BER enzymes the Arabidopsis is dictated5-methylcytosine by both the opposite (5-meC) base DNA and glycosylase the methylation ROS1 efficientlyprobabil- ityexcises of the T (=5-methyluracil,sequence context. 5-meU) Based buton these only atobservations T:G mismatches, and our and previous also displays results a strong with preferenceARP and FPG for [26], a CG we sequence hypothesized context that [29 the,30 ].probability Therefore, of themethylation specificity at ofthe some sequence BER flankingenzymes the is dictatedAP site and/or by both the the orphan opposite base base on the and opposite the methylation DNA strand probability may influence of the thesequence probability context. that Based an abasic on these site observationsis processed either and our by previousan AP endonuclease results with ARPor an andAP lyase.FPG [26 ], we hypothesized that the probability of methylation at the sequence flanking the AP siteIn this and/or work the, we orphan analysed base the on activity the opposite of plant DNA and strand human may AP influence endonucleases the probability and AP lyasesthat an on abasic DNA site substrates is processed containing either by an an abasic AP endonuclease site opposite or either an AP G lyase. or C in different sequencIn thise contexts work, with we analysed different the methylation activity of probabilities. plant and human AP sites AP opposite endonucleases G are com- and APmon lyases intermediates on DNA substratesduring the containingrepair of deaminated an abasic site C or opposite 5-meC, either whereas G or AP C insites different oppo- sitesequence C arise contexts after spontaneous with different N7 methylation-meG probabilities. or during AP sitesthe repair opposite of oxidized G are com- G [7].mon We intermediates found that during in all the tested repair sequence of deaminated contexts C, orArabidopsis 5-meC, whereas ARP endonuclease AP sites opposite dis- playedC arise a after significantly spontaneous higher N7-meG activity depurination on AP sites oropposite during G. the In repair contrast, of oxidizedFPG AP Glyase [7]. Weshowed found a preference that in all tested for AP sequence sites opposite contexts, C. TheArabidopsis major humanARP endonuclease AP endonuclease displayed (APE1) a prefersignificantlyred G as higher the orphan activity base on in AP some sites sequence opposite contexts, G. In contrast, whereas FPG in the AP AP lyase lyase showed activity a preferencedetected in forhuman AP sites cells opposite extracts C.the The opposite major humanbase dependence AP endonuclease was different (APE1) for preferred β- and β, G δas-elimination the orphan basecatalysts. in some Our sequence results suggest contexts, that whereas plant inAP the endonucleases AP lyase activity and detected AP lyases in performhuman cells complementary extracts the opposite functions base in the dependence maintenance was differentof C:G pairs, for β counteracting- and β,δ-elimination the po- tentialcatalysts. mutagenic Our results consequences suggest that of plant C deamination AP endonucleases and G oxidation, and AP lyases respectively. perform comple- mentary functions in the maintenance of C:G pairs, counteracting the potential mutagenic 2.consequences Results of C deamination and G oxidation, respectively. 2.1.2. Results Design of DNA Substrates 2.1. DesignGenomic of DNA studies Substrates performed in Arabidopsis determined the relationship between se- quenceGenomic context studies and probability performed of inmethylationArabidopsis indetermined the three methylation the relationship contexts between existing se- inquence plants, context CG, CHG and probabilityand CHH [31,32]. of methylation Based on in thesuch three data, methylation we designed contexts 5′-fluorescein existing- labelledin plants, 51 CG,-mer CHG oligonucleotides and CHH [31 with,32]. a Based single onuracil such residue data, we in designedthree sequence 50-fluorescein- contexts withlabelled different 51-mer probabilities oligonucleotides of methylation with a single (Figure uracil 1). Since residue we inwere three also sequence interested contexts in an- withalysing different the effect probabilities of the orphan of methylationbase at AP sites (Figure arising1). from Since C:G we pairs, were alsowe designed interested com- in plementaryanalysing the oligonucleotides effect of the orphan with baseC or atG APopposite sites arisingthe uracil. from Next, C:G pairs,the abasic we designed site was generatedcomplementary by incubation oligonucleotides with Escherichia with C orcoli G Uracil opposite DNA the Glycosylase uracil. Next, (UDG) the abasic (see siteMateri- was alsgenerated and Methods). by incubation Depending with Escherichia on the opposite coli Uracil base, DNA the six Glycosylase different (UDG)DNA substrates (see Materials pre- sentand Methods).different probabilities Depending of on DNA the opposite methylation base, at the the six orphan different cytosine DNA (when substrates the opposite present basedifferent is C) probabilities or the lost cytosine of DNA (when methylation the opposite at the orphanbase is cytosineG) (Supplementary (when the opposite Table S1). base is C) or the lost cytosine (when the opposite base is G) (Supplementary Table S1).

Figure 1. Oligonucleotide duplexesduplexes withwith threethree different different sequence sequence contexts contexts and and either either G orG or C oppositeC oppo- 0 sitethe APthe siteAP usedsite used as DNA as DNA substrates. substrates. Fluorescein Fluorescein labelling labelling at the 5 atend the of 5′ the end upper of the strand upper is indicatedstrand is indicatedby an asterisk. by an X asterisk. indicates X Gindicates or C. G or C. 2.2. The Main Arabidopsis AP Endonuclease, ARP, Prefers G to C as the Base Opposite the Abasic Site To determine whether the activity of the main Arabidopsis AP endonuclease is in- fluenced by a context sequence with a different preference to be methylated and/or by the base opposite the abasic site, we performed incision assays with recombinant ARP protein and the six different DNA substrates described above. The results obtained show a preference of recombinant ARP in processing AP sites opposite G in comparison to AP:C Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 16

2.2. The Main Arabidopsis AP Endonuclease, ARP, Prefers G to C as the Base Opposite the Abasic Site To determine whether the activity of the main Arabidopsis AP endonuclease is influ- enced by a context sequence with a different preference to be methylated and/or by the base opposite the abasic site, we performed incision assays with recombinant ARP protein and the six different DNA substrates described above. The results obtained show a pref- erence of recombinant ARP in processing AP sites opposite G in comparison to AP:C sites in all three sequence contexts (Figure 2). The preference of ARP for AP:G targets was most evident with context C and the higher substrate concentration, with more than 80% of DNA being processed when the AP site was opposite guanine, but less than 10% when the orphan base was cytosine (Figure 2B). In general, ARP showed a higher activity on AP sites located at B or C contexts, being Int. J. Mol. Sci. 2021, 22, 8763 the A context processed with a lower efficiency (Figure 2). In reactions with 80 nM of AP:G4 of 15 substrates (Figure 2B), a preference of the for sequence context C was observed, reaching 80% of the processed substrate in 30 min, whereas only 20% or 40% substrate was processed with substrates with contexts A or B, respectively. With AP:C targets, lower sites in all three sequence contexts (Figure2). The preference of ARP for AP:G targets was incision efficiencies in the A context were also observed, particularly at the higher DNA most evident with context C and the higher substrate concentration, with more than 80% concentration. Thus, at 180 min and 80 nM DNA, only 55% of substrate with context A of DNA being processed when the AP site was opposite guanine, but less than 10% when was processed, compared to 91% for contexts B or C. the orphan base was cytosine (Figure2B).

FigureFigure 2. 2.EffectEffect of offlanking flanking sequence sequence context context and and orphan orphan base base on on the the AP AP endonuclease endonuclease activity activity of of recombinantrecombinant ArabidopsisArabidopsis ARP.ARP. His His-ARP-ARP protein protein (10 (10 nM) nM) was was incubated incubated at at37 37 °C◦ Cwith with 40 40 nM nM (A ()A or) or 8080 nM nM (B ()B DNA) DNA substrates substrates (contexts (contexts A, A, B Bor or C) C) containing containing an an AP AP site site opposite opposite guanine guanine (orange) (orange) or or cytosine (blue). After stabilization with NaBH4, reaction products were separated by denaturing cytosine (blue). After stabilization with NaBH4, reaction products were separated by denaturing PAGE,PAGE, detected detected by by fluorescence fluorescence scanning andand quantified.quantified. Data Data are are the the mean mean and and standard standard error error from from three independent experiments. three independent experiments.

WeIn next general, analysed ARP showedthe effect a higherof the activitysequence on context AP sites and located orphan at B base or C on contexts, the native being ARPthe activity A context detected processed in plant with awhole lower-cell efficiency extracts. (Figure To exclude2). In reactions AP lyase with activity, 80 nM we of used AP:G fpgsubstrates−/− mutant (Figureplants. 2TheB), acell preference extract quality of the and enzyme DNA for repair sequence competence context were C was previously observed, verifiedreaching by 80%measuring of the processeduracil DNA substrate glycosylase in 30 activity min, whereas in comparison only 20% with or 40%WT extracts substrate onwas a DNA processed duplex with containing substrates uracil with (Figure contexts 3A). A orWe B, then respectively. examined With the AP:Clevel of targets, AP endo- lower nucleaseincision and efficiencies AP lyase in activities the A context in cell wereextracts also from observed, WT, arp particularly−/− and fpg−/− atplants the higher on a DNA DNA substrateconcentration. containing Thus, an atAP 180 site min (Figure and 803B). nM The DNA, Mg2+- onlydependent 55% of AP substrate incision with activity context was A was processed, compared to 91% for contexts B or C. We next analysed the effect of the sequence context and orphan base on the native ARP activity detected in plant whole-cell extracts. To exclude AP lyase activity, we used

fpg−/− mutant plants. The cell extract quality and DNA repair competence were previously verified by measuring uracil DNA glycosylase activity in comparison with WT extracts on a DNA duplex containing uracil (Figure3A). We then examined the level of AP endonuclease and AP lyase activities in cell extracts from WT, arp−/− and fpg−/− plants on a DNA substrate containing an AP site (Figure3B). The Mg 2+-dependent AP incision activity was lost in arp−/− mutants. In contrast, AP incision levels obtained with fpg−/− extracts were comparable to those of WT extracts, but only in the presence of Mg2+; thus, demonstrating that the only AP processing capacity detected in fpg−/− extracts was AP endonuclease activity from ARP. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 16

lost in arp−/− mutants. In contrast, AP incision levels obtained with fpg−/− extracts were com- parable to those of WT extracts, but only in the presence of Mg2+; thus, demonstrating that Int. J. Mol. Sci. 2021, 22, 8763 5 of 15 the only AP processing capacity detected in fpg−/− extracts was AP endonuclease activity from ARP.

−/− −/− FigureFigure 3. Incision 3. Incision assay assay for characterization for characterization of Arabidopsis of Arabidopsis WT, arpWT,−/− andarp fpg−/− celland extracts.fpg cell(A) Uracil extracts. − − − − DNA(A) glycosylase Uracil DNA activity. glycosylase Cell extracts activity. from Cell WT, extracts arp−/− from and WT,fpg−/−arp lines/ (25and µg)fpg were/ lines incubated (25 µg) for were 3 h atincubated 37 °C in forthe 3presence h at 37 ◦ ofC in20 thenM presence of DNA ofsubstrate 20 nM ofcontaining DNA substrate an uracil. containing Reaction an products uracil. Reaction were separatedproducts by were denaturing separated PAGE bydenaturing and detected PAGE by fluorescence and detected scanning. by fluorescence M, 28 size scanning. marker M,with 28 3′ size- −/− −/− OH.marker (B) AP with lyase 30 -OH.and AP (B) endonuclease AP lyase and activity. AP endonuclease WT, arp activity. and fpg WT, cellarp extracts−/− and (25fpg µg)−/− werecell extractsincu- bated(25 µforg) 3 were h at 37 incubated °C in the for presence 3 h at 37 of◦ 20C innM the of presenceDNA substrate of 20 nM containing of DNA an substrate AP site containingopposite G, an either in the presence or in the absence of 2 mM Mg2+. After stabilization with NaBH4, reaction prod- AP site opposite G, either in the presence or in the absence of 2 mM Mg2+. After stabilization with ucts were separated and detected as indicated above. NaBH4, reaction products were separated and detected as indicated above.

WhenWhen we we analysed analysed the theAP APincision incision activity activity of native of native ARP on ARP the on different the different DNA sub- DNA strates,substrates, we also we observed also observed a clear a clear preference preference for AP for APsites sites opposite opposite guanine guanine (Figure (Figure 4).4 ).In In contrastcontrast to torecombinant recombinant ARP, ARP, curves curves obtained obtained with with the the native native enzyme enzyme were were rectangular rectangular hyperbolas that could be fitted to the equation [Product] = Pmax [1 − exp(−kt)]; thus, (allowing−kt) hyperbolas that could be fitted to the equation [Product] = Pmax [1 − exp ]; thus, theallowing calculation the of calculation kinetic parameters of kinetic (see parameters Materials (see and Materials Methods). and The Methods). relative processing The relative efficiency (Erel) of native ARP on AP:G was significantly higher than on AP:C in all three processing efficiency (Erel) of native ARP on AP:G was significantly higher than on AP:C sequencein all three contexts sequence (Supplementary contexts (Supplementary Table S2). However, Table the S2). prefere However,nce for the G preference as the orphan for G baseas was the orphanless noticeable base was in lesscontext noticeable B. As a inresult, context for AP B. As sites a result,opposite for guanine AP sites, a opposite lower rel E guanine, was detected a lower in Econtextrel was B detected (1.38 ± 0.02) in context compared B (1.38 to ±contexts0.02) compared A and C to(3.10 contexts ± 0.22 Aand and 2.91C (3.10± 0.02,± respectively).0.22 and 2.91 Conversely,± 0.02, respectively). for AP sites Conversely, opposite cytosine for AP sites, the E oppositerel on context cytosine, B wasthe higher Erel on (0.75 context ± 0.01) B than was that higher observed (0.75 ± in0.01) contexts than A that and observedC (0.19 ± 0.02 in contexts and 0.38 A± 0.02, and C respectively).(0.19 ± 0.02 Altogether, and 0.38 ± 0.02,these respectively).results indicate Altogether, that ARP, thesethe main results AP indicateendonuclease that ARP, from the Arabidopsismain AP, endonucleasedisplays a significant from Arabidopsis preference, displays for abasic a significantsites opposite preference guanine, for and abasic that sitesits enzymaticopposite activity guanine, is andmodulated that its by enzymatic the specific activity sequence is modulated flanking the by thelesion. specific sequence Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 16 flanking the lesion.

Figure 4. Effect 4. Effectof sequence of context sequence and base context opposite the and abasic base site on opposite the AP endonucleas the abasice activ- site on the AP endonuclease ity of native ARP. Cell extracts from fpg−/− plants (10 μg) were incubated at 37 °C with 20 nM DNA −/− ◦ substratesactivity (contexts of native A, B ARP.or C) containing Cell extracts an AP site from oppositefpg guanineplants (orange) (10 or cytosineµg) were (blue). incubated at 37 C with 20 nM AfterDNA stabilization substrates with NaBH (contexts4, the reaction A,B produ or C)cts were containing separated by an denaturing AP site PAGE, opposite detected guanine (orange) or cytosine by fluorescence scanning and quantified. Data are the mean and standard error from three inde- pendent(blue). experiments. After stabilization with NaBH4, the reaction products were separated by denaturing PAGE, 2.3.detected The Main by Arabidopsis fluorescence AP Lyase, scanning FPG, Prefers and G to quantified. C as the Base Opposite Data are the theAbasic mean Site and standard error from three independentWe next investigated experiments. whether FPG, the main AP lyase detected in Arabidopsis cell extracts [26], is also affected by the sequence context and/or the base opposite the AP site. We performed incision assays with recombinant FPG protein on the different DNA sub- strates, and the results obtained indicate a clear and consistent preference for AP sites opposite cytosine in all three sequence contexts (Figure 5). In general, the AP lyase activity of FPG was lower at higher DNA concentrations, regardless of the opposite base or the sequence context, suggesting a possible inhibition by substrate. As a result, the preference for C as the orphan base was stronger at the low DNA concentration (Figure 5A). For example, in context C (20 nM), FPG processed about 100% of the substrate containing AP:C after 90 min, compared to 20% substrate with AP:G. The AP lyase activity of FPG on its preferred target (AP:C) was less efficient in con- text A compared to contexts B and C. Thus, for 40 nM substrate, only 30% of AP:C lesions were processed at 90 min, in contrast with 70% or 80% in contexts B and C, respectively (Figure 5B). In these same conditions, no clear differences between contexts were detected for AP:G targets.

Int. J. Mol. Sci. 2021, 22, 8763 6 of 15

2.3. The Main Arabidopsis AP Lyase, FPG, Prefers G to C as the Base Opposite the Abasic Site We next investigated whether FPG, the main AP lyase detected in Arabidopsis cell extracts [26], is also affected by the sequence context and/or the base opposite the AP site. We performed incision assays with recombinant FPG protein on the different DNA substrates, and the results obtained indicate a clear and consistent preference for AP sites opposite cytosine in all three sequence contexts (Figure5). In general, the AP lyase activity of FPG was lower at higher DNA concentrations, regardless of the opposite base or the sequence context, suggesting a possible inhibition by substrate. As a result, the preference Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWfor C as the orphan base was stronger at the low DNA concentration (Figure5A).7 of For 16 example, in context C (20 nM), FPG processed about 100% of the substrate containing AP:C after 90 min, compared to 20% substrate with AP:G.

FigureFigure 5. 5.EffectEffect of ofsequence sequence context context and and base base opposite opposite the the abasic abasic site site on on the the AP AP lyase lyase activity activity of of ◦ recombinantrecombinant FPG. FPG. His His-FPG-FPG protein protein (2 (2nM) nM) was was incubated incubated at at37 37 °C Cwith with 20 20 nM nM (A ()A or) or 40 40 nM nM (B ()B of) of DNADNA substrates substrates (contexts (contexts A, A, B Bor or C) C) containing containing an an AP AP site site opposite opposite guanine guanine (orange) (orange) or or cytosine cytosine (blue).(blue). After After stabilization stabilization with with NaBH NaBH4, reaction4, reaction products products were were separated separated by denaturing by denaturing PAGE, PAGE, de- tecteddetected by fluorescence by fluorescence scanning scanning and and quantified. quantified. Data Data are arethe the mean mean and and standard standard error error from from three three independentindependent experiments. experiments.

WeThe also AP analysed lyase activity the AP of lyase FPG on activity its preferred of the native target (AP:C)FPG enzyme was less using efficient cells inextracts context fromA compared ARP deficient to contexts plants. BAs and shown C. Thus, in Figure for 40 3, nMwe corro substrate,borated only that 30% arp−/− of extracts AP:C lesions had similarwere processedquality and at efficiency 90 min, in in contrast uracil DNA with repair 70% orthan 80% WT in and contexts fpg−/− extracts B and C, (Figure respectively 3A), and(Figure then5 analysedB). In these AP sameendonuclease conditions, and no AP clear lyase differences activity using between an contextsAP site generated were detected by uracilfor AP:G excision targets. (Figure 3B). As expected, AP incision levels of arp−/− extracts were similar to thoseWe of also WTanalysed extracts, thebut APonly lyase in the activity absence of theof Mg native2+. Such FPG Mg enzyme2+-independent using cells activity extracts wasfrom lost ARP in fpg deficient−/− extracts, plants. demonstrating As shown in that Figure arp3−/−, we-deficient corroborated plants thatonly arppossess−/− extracts AP lyase had activitysimilar from quality FPG. and efficiency in uracil DNA repair than WT and fpg−/− extracts (Figure3A), andWe then then analysed performed AP endonuclease AP incision assays and AP with lyase arp activity−/− extracts using and an different AP site generated DNA sub- by strates.uracil Similarly excision (Figure, with 3recombinantB). As expected, FPG, AP the incision results levelsobtained of arp with−/− nativeextracts FPG were also similar re- vealedto those a higher of WT efficiency extracts, butprocessing only in AP the sites absence opposite of Mg cytosine2+. Such (Figure Mg2+-independent 6). This preferenc activitye − − − − waswas more lost evident in fpg / withextracts, context demonstrating B, in which 60% that ofarp the /substrate-deficient was plants processed only possessin 90 min AP whenlyase the activity opposite from base FPG. was cytosine, while only 20% was processed when the base op- −/− posite Wethe thenAP site performed was guanine AP incision(Figure assays6). With with contextarp A, aextracts preference and for different cytosine DNA as the sub- opposingstrates. Similarly,base was withalso recombinantobserved, although FPG, the less results marked obtained (Figure with 6). native Unlike FPG the also results revealed ob- taineda higher with efficiency contexts A processing and B, with AP context sites opposite C we observed cytosine the (Figure lowest6). levels This of preference incision and was no effect depending on the base opposite the AP site (Figure 6). As for the effect of the sequence context on the AP lyase activity of native FPG, we observed greater differences when the AP site was opposite cytosine. A comparison of Erel values for AP:C substrates revealed a preference for contexts A and B (Erel 0.23 ± 0.01 and 0.32 ± 0.01, respectively) compared to context C (0.07 ± 0.00) (Supplementary Table S2). In contrast, with AP:G tar- gets we observed a preference for context A (0.14 ± 0.01) compared to context C (0.10 ± 0.00) (no reliable Erel value could be estimated for context B) (Supplementary Table S2). Altogether, these results indicate that the main AP lyase from Arabidopsis exhibits a pref- erence for abasic sites opposite cytosine, and that its enzymatic activity is modulated by the specific sequence flanking the lesion.

Int. J. Mol. Sci. 2021, 22, 8763 7 of 15

more evident with context B, in which 60% of the substrate was processed in 90 min when the opposite base was cytosine, while only 20% was processed when the base opposite the AP site was guanine (Figure6). With context A, a preference for cytosine as the opposing base was also observed, although less marked (Figure6). Unlike the results obtained with contexts A and B, with context C we observed the lowest levels of incision and no effect depending on the base opposite the AP site (Figure6). As for the effect of the sequence context on the AP lyase activity of native FPG, we observed greater differences when the AP site was opposite cytosine. A comparison of Erel values for AP:C substrates revealed a preference for contexts A and B (Erel 0.23 ± 0.01 and 0.32 ± 0.01, respectively) compared to context C (0.07 ± 0.00) (Supplementary Table S2). In contrast, with AP:G targets we observed a preference for context A (0.14 ± 0.01) compared to context C (0.10 ± 0.00) (no reliable Erel value could be estimated for context B) (Supplementary Table S2). Altogether, these results indicate that the main AP lyase from Arabidopsis exhibits a preference for Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 16 abasic sites opposite cytosine, and that its enzymatic activity is modulated by the specific sequence flanking the lesion.

FigureFigure 6. EffectEffect of of sequence sequence context context and and base base opposite opposite the the abasic abasic site site on on the AP lyase activity of − − ◦ nativenative FPG. FPG. Cell Cell extracts extracts from from arparp−/− plants/ plants (25 (25μg) µwereg) were incubated incubated at 37 at °C 37 withC with20 nM 20 DNA nM DNA sub- stratessubstrates (contexts (contexts A, B A,or BC) or containing C) containing an AP an site AP opposite site opposite guanine guanine (orange) (orange) or cytosine or cytosine (blue). (blue). After

stabilizationAfter stabilization with NaBH with4 NaBH, the reaction4, the reaction products products were separated were separated by denaturing by denaturing PAGE, detected PAGE, de-by fluorescencetected by fluorescence scanning and scanning quantified. and quantified.Data are the Data mean are and the standard mean and error standard from thr erroree independent from three experiments.independent experiments.

2.4.2.4. The The Major Major Human Human AP AP Endonuclease, Endonuclease, APE1, APE1, Exhibits a Preference for G as the Orphan Base, butbut Not Not in in All All Sequence Sequence Contexts Next,Next, we we wondered if the inverse preference for the orphan base exhibited by plant AP endonucleases and AP lyases and the modulatory effect exerted on their activities by thethe flanking flanking DNA DNA sequence sequence were were common common features features present present in in other organisms such as humans.humans. We We first first analysed the enzymatic activity of recombinant and native APE1, the majormajor human AP endonu endonucleaseclease [33], [33], on the different DNA substrates.substrates. The recombinant version of human human APE1 showed showed a slight slight preference preference for AP sites opposite opposite guanine but only only in sequence context B (E 8.03 ± 0.06 and 4.67 ± 0.05 for AP:G and AP:C, respectively) in sequence context B (Erelrel 8.03 ± 0.06 and 4.67 ± 0.05 for AP:G and AP:C, respectively) (Figure(Figure 77,, SupplementarySupplementary TableTable S3).S3). RegardingRegarding thethe contextcontext sequence,sequence, andand irrespectiveirrespective ofof thethe base base opposite opposite the the abasic abasic site, site, the the highest highest processing processing activity activity was was observed observed with with context context A (with Erel values close to 9), while the lowest was observed with context C (with Erel A (with Erel values close to 9), while the lowest was observed with context C (with Erel values close to 5) (Figure7). values close to 5) (Figure 7).

Figure 7. Effect of sequence context and base opposite the abasic site on the AP endonuclease activ- ity of recombinant human APE1. APE1 protein (4 nM) was incubated at 37 °C with 80 nM DNA substrates (contexts A, B or C) containing an AP site opposite guanine (orange) or cytosine (blue). After stabilization with NaBH4, reaction products were separated by denaturing PAGE, detected by fluorescence scanning and quantified. Data are the mean and standard error from three independent experiments.

We also analysed the activity of native human APE1 using U2OS osteosarcoma cell extracts. The results obtained were very similar to those obtained with recombinant APE1

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 16

Figure 6. Effect of sequence context and base opposite the abasic site on the AP lyase activity of native FPG. Cell extracts from arp−/− plants (25 μg) were incubated at 37 °C with 20 nM DNA sub- strates (contexts A, B or C) containing an AP site opposite guanine (orange) or cytosine (blue). After stabilization with NaBH4, the reaction products were separated by denaturing PAGE, detected by fluorescence scanning and quantified. Data are the mean and standard error from three independent experiments.

2.4. The Major Human AP Endonuclease, APE1, Exhibits a Preference for G as the Orphan Base, but Not in All Sequence Contexts Next, we wondered if the inverse preference for the orphan base exhibited by plant AP endonucleases and AP lyases and the modulatory effect exerted on their activities by the flanking DNA sequence were common features present in other organisms such as humans. We first analysed the enzymatic activity of recombinant and native APE1, the major human AP endonuclease [33], on the different DNA substrates. The recombinant version of human APE1 showed a slight preference for AP sites opposite guanine but only in sequence context B (Erel 8.03 ± 0.06 and 4.67 ± 0.05 for AP:G and AP:C, respectively) (Figure 7, Supplementary Table S3). Regarding the context sequence, and irrespective of the base opposite the abasic site, the highest processing activity was observed with context Int. J. Mol. Sci. 2021, 22, 8763 8 of 15 A (with Erel values close to 9), while the lowest was observed with context C (with Erel values close to 5) (Figure 7).

FigureFigure 7. 7. EffectEffect of ofsequence sequence context context and and base base opposite opposite the abasic the abasic site on site the on AP the endonuclease AP endonuclease activ- ◦ ityactivity of recombinant of recombinant human human APE1. APE1. APE1 APE1protein protein (4 nM) (4 was nM) incubated was incubated at 37 °C at with 37 C 80 with nM 80DNA nM substratesDNA substrates (contexts (contexts A, B orA, C)B conta or C)ining containing an AP site anAP opposite site opposite guanine guanine (orange) (orange) or cytosine or cytosine (blue). After(blue). stabilization After stabilization with NaBH with4, reaction NaBH4 products, reaction were products separated were by separated denaturing by PAGE, denaturing detected PAGE, by Int. J. Mol. Sci. 2021fluorescencedetected, 22, x FOR by PEER fluorescencescanning REVIEW and scanning quantified. and Data quantified. are the mean Data and arethe standard mean anderror standard from three error independen from threet 9 of 16 experiments.independent experiments.

WeWe also also analysed analysed the the activity activity of of native native human human APE1 APE1 using using U2OS U2OS osteosarcoma osteosarcoma cell cell extracts.extracts. The The results results(Figure obtained obtained 8), suggesting were were very very that similar similar most, toif to notthose those all, obtained obtainedof the native with with APrecombinant recombinant endonucle APE1ase APE1 activity detected (Figure8), suggestingin these that cell most, extracts if not came all, from of the APE1. native However, AP endonuclease in this case activity, the preference detected for orphan G in these cell extractswas also came marginally from APE1. detectable However, in incontexts this case, A and the preferenceC (Figure 8 for and orphan Supplementary G Table was also marginallyS3). detectable in contexts A and C (Figure8 and Supplementary Table S3).

Figure 8. EffectFigure of the 8. Effectsequence of thecontext sequence and base context opposite and baseto the opposite abasic site to on the the abasic native site AP on endonuclease the native AP activity. Cell ◦ extracts (0.1endonuclease μg) from U2OS activity. were incubated Cell extracts at 37 (0.1 °Cµ g)with from 20 U2OSnM DNA were substrates incubated (contexts at 37 C A, with B or 20 C). nM After DNA stabilization with NaBHsubstrates4, the reaction (contexts products A, B were or C). separated After stabilization by denaturing with NaBH PAGE,4, thedetected reaction by productsfluorescence were scanning separated and quanti- fied. Data areby the denaturing mean and PAGE, standard detected error from by fluorescence three independent scanning experiments. and quantified. Data are the mean and standard error from three independent experiments. We next examined the native AP lyase activity in U2OS cell extracts. Unlike plant We next examinedextracts, human the native extracts AP lyaseexhibit activityed the activi in U2OSty of cell several extracts. AP lyases, Unlike some plant of which gener- extracts, humanate extractsd 3′-PUA exhibited ends (NTH1 the activity and OGG1) of several and AP others lyases, produce somed of 3 which′-P termini gener- (NEIL1, NEIL2 0 0 ated 3 -PUA endsand (NTH1 NEIL3) and [34]. OGG1) We, therefore and others, quantitated produced 3both-P termini types of (NEIL1, DNA repair NEIL2 intermediates and in the NEIL3) [34]. We,three therefore, different quantitated sequence both contexts. types ofWe DNA foun repaird that intermediates the accumulation in the of three 3′-PUA ends was 0 different sequencefaster contexts. for AP:G We than found for thatAP:C the targets, accumulation whereasof the 3 -PUAreverse ends was was observed faster for 3′-P ends 0 for AP:G than for(Figure AP:C 9). targets, These whereasresults suggest the reverse that washuman observed β-elimination for 3 -P endsand β,(Figure δ-elimination9). catalysts These results suggestprefer G that and human C, respectively,β-elimination as the and orphanβ, δ-elimination base. catalysts prefer G and C, respectively, as the orphan base.

Figure 9. Effect of sequence context and base opposite the abasic site on the native AP lyase activity detected in human cells. Extracts from U2OS cells (20 μg) were incubated at 37 °C with 20 nM DNA substrates (context A, B or C) containing an AP site opposite guanine (orange) or cytosine (blue).

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 16

(Figure 8), suggesting that most, if not all, of the native AP endonuclease activity detected in these cell extracts came from APE1. However, in this case, the preference for orphan G was also marginally detectable in contexts A and C (Figure 8 and Supplementary Table S3).

Figure 8. Effect of the sequence context and base opposite to the abasic site on the native AP endonuclease activity. Cell extracts (0.1 μg) from U2OS were incubated at 37 °C with 20 nM DNA substrates (contexts A, B or C). After stabilization with NaBH4, the reaction products were separated by denaturing PAGE, detected by fluorescence scanning and quanti- fied. Data are the mean and standard error from three independent experiments.

We next examined the native AP lyase activity in U2OS cell extracts. Unlike plant extracts, human extracts exhibited the activity of several AP lyases, some of which gener- ated 3′-PUA ends (NTH1 and OGG1) and others produced 3′-P termini (NEIL1, NEIL2 and NEIL3) [34]. We, therefore, quantitated both types of DNA repair intermediates in the three different sequence contexts. We found that the accumulation of 3′-PUA ends was faster for AP:G than for AP:C targets, whereas the reverse was observed for 3′-P ends Int. J. Mol. Sci. 2021, 22, 8763 (Figure 9). These results suggest that human β-elimination and β, δ-elimination catalysts9 of 15 prefer G and C, respectively, as the orphan base.

FigureFigure 9. 9.EffectEffect of ofsequence sequence context context and and base base opposite opposite the the abasic abasic site site on on the the native native AP AP lyase lyase activity activity detecteddetected in inhuma humann cells. cells. Extracts Extracts from from U2OS U2OS cells cells (20 (20 μg)µg) were were incubated incubated at at 37 37 °C◦ Cwith with 20 20 nM nM DNA DNA substratessubstrates (context (context A, A, B Bor or C) C) containing containing an an AP AP site site opposite opposite guanine guanine (orange) (orange) or or cytosine cytosine (blue). (blue).

After stabilization with NaBH4, the reaction products were separated denaturing PAGE, detected by fluorescence scanning and quantified. Graphs show percentage of incised products with 30-PUA ends (A) or 30-P ends (B). Data are the mean and standard error from three independent experiments.

3. Discussion All cellular organisms possess both AP endonucleases and AP lyases, two different enzymatic activities able to incise abasic sites, but the factors explaining such an apparently redundant role remain poorly understood. In this work, we tested the hypothesis that the specific sequence surrounding an AP site and/or the identity of the orphan base influences the enzymatic activities of AP endonucleases and AP lyases. We concentrated our study on AP sites arising from C:G pairs located within sequence contexts with different probabilities to be targeted by the DNA methylation machinery. Although context-dependent excision has been reported for several DNA glycosy- lases [35], the effect of the flanking DNA sequence on AP incision remains unexplored. In this work, we found significant differences in AP site incision efficiency by both AP endonucleases and AP lyases in different sequence contexts. However, there was no clear correlation between AP incision efficiency and the expected probability of methylation at the C:G pair in which the AP site arose. For example, the native ARP endonuclease activity on AP:G targets was equally efficient in contexts A and C, in which the expected probability of the lost cytosine to be methylated is low and high, respectively. In comparison, it was significantly lower in context B, with an intermediate probability of methylation. Similarly, the native AP lyase activity of FPG on AP:C targets was significantly higher in context A compared to context C, although the probability of the orphan cytosine to be methylated is very similar in both sequences. Likewise, no correlation with DNA methylation probability was found either for human AP endonuclease or AP lyase activities. These results suggest that the capacity of AP endonucleases or AP lyses to incise an AP site arising at a C:G pair is not related to the probability of such a pair to be epigenetically modified. Apparently, the sequence context preference shown by some DNA glycosylases excising frequent lesions arising at CpG sites, such as mismatched U or T [28–30], is not present in downstream steps in the BER pathway, such as AP incision. In any case, the methylation-independent differences in AP processing efficiency de- tected between different sequence contexts suggests that the specific sequence surrounding an abasic site influences the efficiency of AP endonucleases and AP lyases. Systematic Int. J. Mol. Sci. 2021, 22, 8763 10 of 15

studies with a large set of DNA substrates will be needed to identify which sequence features influence AP endonuclease and AP lyase activity and the mechanism involved. An important finding arising from our study was the reverse preference of ARP endonuclease and FPG lyase for the orphan base at AP sites originated from C:G pairs. Whereas ARP favoured G as the estranged base, FPG displayed a preference for abasic sites opposite C. The preference of FPG for AP sites opposite C is in agreement with our previous study showing that this enzyme is critical for the excision of AP sites arising from the spontaneous loss of N7-meG [26]. In the present work, the preference of FPG lyase activity for AP:C targets was observed with the recombinant enzyme in all tested sequence contexts, whereas with the native activity in cells extracts it was detected in contexts A and B, but not C. One possible explanation for such a discrepancy is that interacting protein partners present in the cell extract modulate the orphan base preference of FPG in a sequence-dependent manner. On the other hand, it is worth noting that the recombinant protein used in our study is one (FPG1) of seven potential isoforms generated by the alternative splicing of the FPG primary transcript [36]. FPG1 mRNA is expressed in flowers and roots but poorly in leaves, which were the primary material used in our cell extracts. Future work will be needed to analyse whether the preference for AP:C targets is conserved in all FPG isoforms. Proteins from the Fpg subfamily are bifunctional DNA glycosylases involved in oxoG repair in both prokaryotes [37] and plants [38]. Given their role in removing oxidized guanine, a preference for C in the complementary strand is not unexpected. However, there are conflicting reports on the efficiency of bacterial Fpg excising oxoG opposite C or G. Thus, one study reported that E. coli Fpg processes oxoG:C about 5-fold faster than oxoG:G [39], whereas another report showed about a 10-fold higher activity on oxoG:G than on oxoG:C [40]. On the other hand, E. coli Fpg excises N(4),5-dimethylcytosine opposite C, but not opposite G [41]. To our knowledge, no data on the AP lyase activity of prokaryotic Fpg on DNA substrates with different opposite bases have been published. Unlike plant extracts, in which the only detectable AP lyase activity is that of FPG [26], human cell extracts likely contain a mixture of different AP lyases, making it difficult to detect specific preferences for the orphan base. There are five human bifunctional DNA glycosylases with AP lyase activity NTH1 and OGG1 are β-elimination catalysts and, therefore, generate 30-PUA ends, whereas NEIL1, NEIL2 and NEIL3 are β, δ-elimination catalysts and generate 30-P ends [34]. We found that the accumulation of 30-PUA ends generated by the AP lyase activity of human cell extracts was higher for AP:G than for AP:C targets, while the inverse situation was detected for 30-P ends. Interestingly, it has been reported that human NTH1 preferentially incises AP sites opposite G [42], which might partially explain the higher accumulation of 30-PUA incision products that we have detected for AP:G targets. The most likely candidates for the detected 30-ends are NEIL1, NEIL2 and/or NEIL3, but their opposite base preference as AP lyases, if any, remains unknown. Conversely, to the preference for orphan C of Arabidopsis FPG AP lyase, we found that ARP endonuclease displayed a significantly higher activity on AP sites opposite G. Whereas the preference of recombinant ARP for AP:G over AP:C was similar among all three sequence contexts, the native ARP activity detected in plant cell extracts showed between a 2-fold and 15-fold higher efficiency on AP:G than on AP:C targets, depending on the specific sequence context. Such a preference for G as the orphan base might partially explain the very poor activity of ARP on AP sites arising from the spontaneous loss of N7-meG [26]. Interestingly, and in agreement with our previous results [26], we found that human APE1 endonuclease also displayed some preference for AP sites opposite G, particularly in reactions catalysed by the native AP endonuclease activity detected in cell extracts. Thus, native APE1 displayed between a 1.3-fold and 2.3-fold higher efficiency on AP:G than on AP:C targets, depending on the specific sequence context. The modulatory effect exerted by the sequence flanking the AP site on the opposite base dependence is apparently different for plant and human AP endonucleases. Thus, for ARP, the preference for orphan G was Int. J. Mol. Sci. 2021, 22, 8763 11 of 15

more evident in contexts A and C, but for human APE1 such a preference was greater in context B. Conflicting results have been previously published on opposite base effects on APE1 activity. Some studies did not find any detectable influence of the orphan base [43], but others reported a preference for a opposite the AP site [44]. Interestingly, one study found that AP sites opposite G were repaired 1.2–4.7-fold more efficiently than AP-sites opposite A in five out of eight different human cell extracts [45]. It is possible that the modulatory effects of the flanking sequence might partially explain such disparities. Our results suggest that FPG and ARP specifically interacts with the base opposite the AP site. Interestingly, a structural study with Lactococcus lactis Fpg in a complex with an AP site analogue proposed that a as the orphan base contributes to the optimal conformation of the substrate through specific interactions with an Arg residue that is con- served in Arabidopsis FPG [46]. Therefore, conserved structural features in Fpg homologs may explain the preference for AP sites opposite C that we have observed with FPG. It is also possible that FPG and/or ARP discriminate between conformational differ- ences in AP sites opposite different bases. For example, NMR studies have revealed that the ratio of the different anomeric forms in which an AP site exists may depend upon the identity of the opposite base in the complementary strand [47]. Altogether, our results suggest that, at least in plants, AP endonucleases and AP lyases perform complementary roles in processing AP sites arising at C:G pairs. We propose a model (Supplementary Figure S1) in which FPG AP lyase preferentially targets AP sites arising from a lost guanine that has been either spontaneously released, frequently after alkylation damage, or enzymatically excised, commonly after oxidation damage. In contrast, ARP endonuclease favours AP sites originating from a missing cytosine, most of which arise during BER of uracil. Future studies are needed to determine whether a similar distribution or roles is observed in other organisms and could be related to the evolutionary origin of AP endonucleases and AP lyases.

4. Materials and Methods 4.1. Plant Material and Cell Extract Preparation Arabidopsis arp−/− (SALK_021478) and fpg−/− (SALK_076932) lines were previously described [26,48]. WT (Col-0) and mutant Arabidopsis seeds were surface sterilized and plated on 10 cm Petri dishes containing 25 mL of 0.44% (w/v) Murashige and Skoog (MS) medium (Sigma, St. Louis, MO, USA) supplemented with 2% (w/v) sucrose and 0.8% (w/v) agar, pH 5.8. Plates were incubated under long-day conditions in a growth chamber at 23 ◦C. After 15 days, seedlings were collected, frozen in liquid nitrogen and stored at 80 ◦C until use. Whole cell extract were prepared as described [49].

4.2. Cell Culture and Cell Extract Preparation Human Bone Osteosarcoma Epithelial Cells, U2OS, purchased from ATCC (U-2 OS, ATCC® HTB96™), were grown in McCoy’s 5a Medium (Biowest, Nuaillé, France), sup- plemented with 10% foetal bovine serum (FBS, Biowest) and 1% penicillin/streptomycin ◦ (Sigma). Cells were maintained in a humidified atmosphere at 37 C and 5% CO2. After cultures became 80% confluent (usually 4 days), cells were trypsinized and counted. Cell pellets (6 × 106 cells) were collected by centrifugation, washed twice in PBS (Sigma, St. Louis, MO, USA), frozen in liquid nitrogen and stored at −80 ◦C until use. Cell pellets were resuspended in lysis buffer (20 mM Tris HCl pH 7.4, 60 mM NaCl, 1 mM DTT, 10% glycerol, 10 µL/mL protease inhibitor Calbiochem® Protease Inhibitor Cocktail Set III, Animal-Free) and lysed by sonication during 3 pulses of 15 s (Bioruptor, Diagenode, Seraing, Belgium). Subsequently, they were centrifuged at 14,000 rpm at 4 ◦C during 8 min and the supernatant was dialyzed at 4 ◦C against dialysis buffer (20 mM Tris HCl pH 7.4, 60 mM NaCl, 1 mM EDTA, 1 mM DTT, 17% glycerol) during 2 h and, after Int. J. Mol. Sci. 2021, 22, 8763 12 of 15

buffer change, dialysis continued during 16 h more. Protein concentration was determined by the Bradford assay [50] and the extract was stored in small aliquots at −80 ◦C.

4.3. Protein Expression and Purification His-ARP and His-FPG were expressed and purified as previously described [26,51].

4.4. Reagents and Enzymes UDG and human AP endonuclease APE1 were obtained from New England Biolabs (NEB).

4.5. DNA Substrates Oligonucleotides used as DNA substrates (Table1) were synthesized by IDT and purified by PAGE before use. Double-stranded DNA substrates were prepared by mixing a 5 µM solution of a 50-fluorescein (Fl)-labelled oligonucleotide with a 10 µM solution of an unlabelled complementary oligonucleotide. Annealing reactions were carried out by heating at 95 ◦C for 5 min followed by slowly cooling to room temperature.

Table 1. Oligonucleotides used as substrates.

Name a DNA Sequence b Strand c Fl_UGCG-F CACGGGATCAATGTGTTCTTTCAGCTCGUGCGTCACGCTGACCAGGAATAC U UGCG-RG GTATTCCTGGTCAGCGTGACGCGCGAGCTGAAAGAACACATTGATCCCGTG L UGCG-RC GTATTCCTGGTCAGCGTGACGCCCGAGCTGAAAGAACACATTGATCCCGTG L Fl_CUCG-F CACGGGATCAATGTGTTCTTTCAGCTCGCUCGTCACGCTGACCAGGAATAC U CUCG-RG GTATTCCTGGTCAGCGTGACGGGCGAGCTGAAAGAACACATTGATCCCGTG L Fl_UCCG-F CACGGGATCAATGTGTTCTTTCAGCTCGUCCGTCACGCTGACCAGGAATAC U UCCG-RG GTATTCCTGGTCAGCGTGACGGGCGAGCTGAAAGAACACATTGATCCCGTG L UCCG-RC GTATTCCTGGTCAGCGTGACGGCCGAGCTGAAAGAACACATTGATCCCGTG L Fl-UGF TCACGGGATCAATGTGTTCTTTCAGCTCUGGTCACGCTGACCAGGAATACC U CGR_G GGTATTCCTGGTCAGCGTGACCGGAGCTGAAAGAACACATTGATCCCGTGA L a Fl, Fluorescein; b, relevant bases are underlined; c U, upper; L, lower.

DNA substrates containing an enzymatic AP site were generated by incubating a DNA duplex containing either a U:G or a U:C mismatch, prepared as described above, with E. coli UDG (0.2 U) at 37 ◦C for 30 min.

4.6. DNA Incision Assay Reactions (50 µL) contained 45 mM Hepes-KOH, pH 7.8, 70 mM KCl, 1 mM DTT, 0.4 mM EDTA, 36 µg BSA, 0.2% glycerol, DNA substrate (20, 40 or 80 nM), the indicated amount of cell extract or protein and, when specified, MgCl2 (2 mM) or EDTA (20 mM). After incubation at 37 ◦C or 30 ◦C, as specified, reactions were stopped at the indicated time by adding 20 mM EDTA, 0.6% SDS and 0.5 mg·mL−1 proteinase K, and mixtures were incubated at 37 ◦C for 30 min. When indicated, reaction products were stabilized by the addition of freshly prepared sodium borohydride (NaBH4; Sigma-Aldrich) to a final concentration of 230 mM and incubated at 0 ◦C for 30 min. The reaction was buffered with 5 µL of Tris-HCl pH 7.5 and 60 µL of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0). As a control, a sample without extract was alkaline treated (NaOH, 15 mM) for 10 min at 70 ◦C to cleave all AP sites present. Subsequently, this sample was buffered with 1.98 µL of Tris-HCl pH 7.5 and 60 µL of TE. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol- precipitated at −20 ◦C in the presence of 0.3 mM NaCl and 16 mg·mL−1 glycogen. Samples were resuspended in 10 µL of 90% formamide and heated at 95 ◦C for 5 min. Reaction products were separated in a 12% denaturing polyacrylamide gel containing 7 M urea. Labelled DNA was visualized using FLA-5100 imager and analysed using Multigauge software (Fujifilm). Int. J. Mol. Sci. 2021, 22, 8763 13 of 15

4.7. Kinetic Analysis We used a previously described model used for the kinetic analysis of human DNA glycosylase (TDG) [52] and the Arabidopsis ROS1 5-methylcytosine DNA glycosy- lase [53]. The product concentration (nM) obtained for the different samples and DNA (−kt) substrates tested were adjusted to the equation [Product] = Pmax [1 − exp ] by a non- linear regression analysis performed with SigmaPlot software. In each case, we determined the parameters Pmax (maximum concentration of processed substrate), T50 (time required to reach 50% of the maximum product, Pmax) and Erel (relative processing efficiency, calculated as Pmax/T50).

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/ijms22168763/s1. Author Contributions: Conceptualization, R.R.A., T.R.-A. and D.C.-C.; methodology, R.R.A., T.R.-A. and D.C.-C.; software, M.J.-R., C.B.-M., M.D.M.-R. and D.C.-C.; validation, M.J.-R., C.B.-M., M.D.M.-R., M.I.M.-M., R.R.A., T.R.-A. and D.C.-C.; formal analysis, M.J.-R., C.B.-M., M.D.M.-R. and D.C.-C.; investigation, M.J.-R., C.B.-M., M.D.M.-R., M.I.M.-M. and D.C.-C.; resources, M.I.M.-M.; data curation, M.J.-R. and D.C.-C.; writing—original draft preparation, R.R.A., T.R.-A. and D.C.-C.; writing—review and editing, M.J.-R., M.I.M.-M., R.R.A., T.R.-A. and D.C.-C.; visualization, M.J.-R. and D.C.-C.; super- vision, R.R.A., T.R.-A. and D.C.-C.; project administration, R.R.A. and T.R.-A.; funding acquisition, T.R.-A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Science and Innovation (MICINN) as well as the European Regional Development Fund (FEDER), under grant number PID2019- 109967GB-I00. M.J.R was supported by a PhD fellowship from the University of Córdoba (Plan Propio de Investigación). Acknowledgments: We are grateful to members of our lab for helpful criticism and advice. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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