Prereplicative repair of oxidized bases in the is mediated by NEIL1 DNA glycosylase together with replication proteins

Muralidhar L. Hegdea,b,1, Pavana M. Hegdea, Larry J. Bellota, Santi M. Mandalc, Tapas K. Hazrac, Guo-Min Lid, Istvan Boldoghe, Alan E. Tomkinsonf, and Sankar Mitraa,1

Departments of aBiochemistry and Molecular Biology, bNeurology, cInternal Medicine, and eMicrobiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555; dGraduate Center for Toxicology and Markey Center, University of Kentucky College of Medicine, Lexington, KY 40536; and fDepartment of Internal Medicine and University of New Mexico Cancer Center, University of New Mexico, Albuquerque, NM 87131

Edited* by Aziz Sancar, University of North Carolina at Chapel Hill, Chapel Hill, NC, and approved July 5, 2013 (received for review March 5, 2013) Base oxidation by endogenous and environmentally induced reactive NEIL2 DNA glycosylases (5, 6) of the Nei family (which also oxygen species preferentially occurs in replicating single-stranded contains the less characterized NEIL3; ref. 7) are distinct from templates in mammalian genomes, warranting prereplicative re- NTH1 and OGG1 of the Nth family because the NEILs can excise pair of the mutagenic base lesions. It is not clear how such lesions damaged bases from ssDNA substrates (8). Furthermore, NEIL1 (which, unlike bulky adducts, do not block replication) are recognized is activated during the S phase (5). Our earlier studies also showed for repair. Furthermore, strand breaks caused by base excision from that NEIL1 functionally interacts with many DNA replication ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would gen- proteins including sliding clamp proliferating cell nuclear antigen erate double-strand breaks during replication, which are not exper- (PCNA), flap endonuclease 1 (FEN-1), and Werner RecQ helicase imentally observed. NEIL1, whose deficiency causes a mutator pheno- (WRN) via its disordered C-terminal segment (9–12). Importantly, type and is activated during the S phase, is present in the DNA mammalian ssDNA-binding replication protein A (RPA), essential replication complex isolated from human cells, with enhanced as- for DNA replication and most other DNA transactions, inhibits sociation with DNA in S-phase cells and colocalization with replica- NEIL1 or NEIL2 activity with primer-template DNA substrates tion foci containing DNA replication proteins. Furthermore, NEIL1 mimicking the replication fork, presumably to prevent double- binds to 5-hydroxyuracil, the oxidative deamination product of C, strand break formation (13). Although they collectively implicate in replication protein A-coated ssDNA template and inhibits DNA NEIL1 in the repair of replicating DNA, those observations did not δ synthesis by DNA polymerase . We postulate that, upon encoun- provide direct evidence for NEIL1’s role in prereplicative repair, tering an oxidized base during replication, NEIL1 initiates prerepli- nor did they address whether NEIL1 is unique for this function. In “ ” cative repair by acting as a cowcatcher and preventing nascent this report, we document that NEIL1 binds to the lesion base in an chain growth. Regression of the stalled replication fork, possibly RPA-coated ssDNA template in vitro, without excising the lesion mediated by annealing helicases, then allows lesion repair in the and cleaving the DNA strand, and blocks primer elongation by the reannealed duplex. This model is supported by our observations replicative DNA polymerase δ (Polδ). This strongly suggests that that NEIL1, whose deficiency slows nascent chain growth in oxi- the replication complex at the lesion site is stalled in vivo in the datively stressed cells, is stimulated by replication proteins in vitro. presence of NEIL1, which provides the signal for repair of lesions in Furthermore, deficiency of the closely related NEIL2 alone does not the template strand before replication. affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of Results NEIL1-mediated prereplicative repair of oxidized bases in the rep- NEIL1 Depletion Inhibits DNA Replication Fork Progression After licating strand, with NEIL2 providing a backup function. Oxidative Stress. Control and NEIL1-depleted (siRNA-mediated) HEK 293 cells (Fig. 1D) were subjected to DNA fiber analysis to genome damage repair | replication fork stalling | oxidized base repair at DNA replication fork Significance everal dozen oxidatively modified, and mostly mutagenic, bases are induced in the genomes of aerobic organisms by Repair of mutagenic oxidized bases in the genome is required S before replication to prevent . It is unknown how endogenous and environmentally induced fl (ROS) (1, 2). For example, 5-hydroxyuracil (5-OHU), a pre- such base lesions, which do not block replication, are agged for repair in the single-stranded replicating template. We dem- dominant lesion generated by oxidative deamination of C, is – mutagenic because of its mispairing with A (3). The bases in the onstrate here that the repair-initiating, S-phase activated Nei- like (NEIL) 1 DNA glycosylase binds to but does not excise the single-stranded (ss) replicating DNA template are particularly base lesion and cleave the template DNA strand, which would prone to oxidation (4); the lack of their repair before replication lead to a lethal double-strand break. Instead, NEIL1 blocks pro- could fix the mutations. The bulky base adducts if formed in the gression of the replication fork, which then regresses to allow template strand would block replication and trigger DNA dam- lesion repair. In the absence of NEIL1, the related glycosylase age-response signaling. In contrast, oxidized bases with minor NEIL2 serves as a backup . modifications, which are continuously formed in much higher

abundance than the bulky adducts, would mostly allow replica- Author contributions: M.L.H., T.K.H., and S.M. designed research; M.L.H., P.M.H., L.J.B., tion. This raises the question of how these bases are marked for S.M.M., and I.B. performed research; G.-M.L. and A.E.T. contributed new reagents/analytic repair before replication to avoid mutagenic consequences. Ox- tools; M.L.H., T.K.H., and S.M. analyzed data; and M.L.H. and S.M. wrote the paper. idized base repair in mammalian genomes occurs primarily via The authors declare no conflict of interest. the (BER) pathway which is initiated with *This Direct Submission article had a prearranged editor. lesion base excision mediated by one of five major DNA gly- 1To whom correspondence may be addressed: [email protected] or [email protected]. cosylases belonging to the Nth or Nei families, with distinct struc- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tural features and reaction mechanisms (1). Nei-like (NEIL) 1 and 1073/pnas.1304231110/-/DCSupplemental.

E3090–E3099 | PNAS | Published online July 29, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1304231110 Downloaded by guest on October 1, 2021 measure DNA replication chain elongation rate after induction of (mouse; Abcam) and NEIL1 or NEIL2 Abs (rabbit) (11, 21). Sec- PNAS PLUS oxidized bases in the genome (14, 15). The cells were sequentially ondary Abs were Alexa Fluor anti-mouse 488 and anti-rabbit 568. treated with chlorodeoxyuridine (CldU), H2O2, and iododeoxyur- Confocal microscopy revealed strong nuclear colocalization of idine (IdU). DNA fibers from lysed cells were spread on micro- NEIL1 with BrdU fluorescence (Fig. 2A). The sliding clamp PCNA, scopic slides, and the progression of replication forks was visualized an S phase marker, served as a positive control. Colocalization of by detecting CldU (red) and IdU (green) tracks by using appro- NEIL2 with BrdU was insignificant in control cells, but significantly priate Abs, as described in Materials and Methods (Fig. 1A). The higher in NEIL1-deficient cells (Fig. 2B). NEIL1 was depleted by double red/green staining represents elongation of preexisting rep- using siRNA as in Fig. 1D. These results indicate strong association lication forks, whereas the red-only and green-only tracks represent of NEIL1 with replicating DNA in WT cells; NEIL2 was associated terminated and newly initiated forks, respectively. Quantification of with replicating DNA only in NEIL1-deficient cells. red and green track lengths in double-labeled DNA showed sig- nificant inhibition of fork progression after H2O2 treatment, as Association of NEIL1 with DNA Replication Proteins Bound to the S-Phase indicated by a higher proportion of shorter IdU tracks in NEIL1- Cell Genome. By using chromatin immunoprecipitation (ChIP)/ depleted cells compared with controls (Fig. 1B). The global rate re-ChIP analysis, we tested whether NEIL1 and DNA replication of fork progression, assessed by measuring the lengths of 100 fork proteins colocalize in the same sequences of the replicating ge- fibers, decreased by ∼20% to 30% in NEIL1-depleted cells under nome. After synchronizing by using double thymidine block treat- oxidative stress (Fig. 1C). In contrast, depletion of NEIL2 did not ment (22), HEK 293 cells were harvested at the G1/S boundary or significantly affect the fork progression rate. However, the in- in the S phase (Fig. 3D)andfixedwith1%formaldehydeforChIP hibition of DNA synthesis in combined (i.e., NEIL1 + NEIL2)- analysis using the NEIL1 immunoprecipitation (IP) with normal depleted cells was markedly higher than that caused by NEIL1 rabbit IgG control. A 250-bp region in the GAPDH or β-actin depletion alone. These results strongly suggest efficient NEIL1- was PCR-amplified (Fig. 3A; primer sequences in Fig. S1B). Sep- mediated repair of oxidized bases in the template DNA whereas arate aliquots of NEIL1-ChIP were subjected to a second IP with repair initiated by other DNA glycosylases including NEIL2 is Abs for PCNA, Polδ, PCNA clamp loader replication factor-C delayed. It is likely that NEIL2 and possibly other DNA glyco- (RF-C),FEN-1,orIgGcontrol.PCR analysis indicated the presence sylases serve as a backup in the absence of NEIL1 (16–18). of these replication proteins bound to the same genomic segment as NEIL1, but only in the S-phase cells. The absence of similar PCR NEIL1 and PCNA Colocalize with the Replication Foci, Whereas NEIL2 products in G1 cells shows that the association of NEIL1 with Colocalizes with PCNA only in NEIL1-Deficient Cells. We analyzed replication proteins was S phase-specific. The lack of amplifica- immunofluorescence to examine colocalization of NEIL1 or tion with control IgG further confirmed the specificity of binding. NEIL2 with BrdU at discrete foci in replicating DNA (19, 20). In a reverse re-ChIP analysis, we first carried out ChIP with RF-C Log-phase HEK 293 cells cultured on microscope coverslips Ab, and the eluate was subjected to a second ChIP with NEIL1, were pulse-labeled with BrdU (10 μM/10 min), fixed with 4% NEIL2 Ab, or IgG control. Again, RF-C and NEIL1 were found (vol/vol) formaldehyde, and then immunostained with anti-BrdU to be bound to the same segments of β-actin and GAPDH in BIOCHEMISTRY

Fig. 1. NEIL1 deficiency inhibits replication fork progression in H2O2-treated human cells. NEIL1- or NEIL2-depleted and control HEK 293 cells in the log phase were sequentially pulse-labeled with CldU (red) and IdU (green) separated by a 10-min treatment with H2O2. DNA fibers were spread on to microscope slides (14, 15) and immunostained with Abs recognizing CldU and IdU (Materials and Methods). Schematic of CldU/IdU labeling (A) and representative immuno- fluorescence images of DNA fibers (B) are shown. (C) Track lengths (in micrometers) of 100 control or NEIL1- or NEIL2-depleted cells were plotted by using ImageJ software. NEIL1- (but not NEIL2-) depleted cells had 20% to 30% inhibition of fork progression, which was increased by simultaneous NEIL2 depletion (∼40%). FEN-1 depletion served asa positive control. (D) More than 75% reductions in NEIL1 and NEIL2 level after siRNA treatment were confirmed by Western analysis of whole cell extracts. The quantitation of protein levels from three separate experiments is represented as a histogram.

Hegde et al. PNAS | Published online July 29, 2013 | E3091 Downloaded by guest on October 1, 2021 92101-KI01; Olink Biosciences). In this assay, two proteins are immunostained with distinct species-specific secondary Abs that are linked to complementary oligonucleotides. When two dif- ferent Ab molecules bind in close proximity (<40 nm), the linked DNA can be amplified and visualized with a fluorescent probe as distinct foci. The assay is highly specific for physically interacting endogenous proteins in a complex (23–25). We detected a large number of nuclear foci with NEIL1–Polδ, NEIL1–RF-C, NEIL1– FEN-1, and NEIL1–LigI pairs (Fig. 4C). No significant signal was observed with control IgG as the primary Ab. Taken together, these results strongly support NEIL1’s functional association with DNA replication proteins, consistent with its preferential role in repairing the replicating genome.

NEIL2’s Enhanced Association with PCNA in NEIL1-Depleted S-Phase Cells Supports Its Backup Role in Prereplicative Repair. We isolated the IP of endogenous PCNA from extracts of G1 or S phase HEK 293 cells by using PCNA Ab (Santa Cruz). Although the NEILs were barely detectable in PCNA IP from G1 cells, the NEIL1 level was markedly higher in the PCNA IP from S phase cells (Fig. 5A), consistent with NEIL1’s higher level in the S phase (5). Furthermore, the NEIL2 level was enhanced ∼10 fold in PCNA IP, normalized to the PCNA level, from extracts of NEIL1-depleted cells (as in Fig. 1D) compared with control siRNA-transfected cells (Fig. 5B). Together, these results show that the PCNA IP contains detectable NEIL1 in S-phase cells, but contains signif- Fig. 2. NEIL1 colocalizes with DNA replication foci. (A) NEIL1 colocalization icant NEIL2 only in NEIL1-depleted cells. This was further with incorporated BrdU (replication foci). (B) NEIL2 colocalization with BrdU is confirmed by in situ PLA assay in HEK 293 cells where a strong observed only in NEIL1-depleted cells. Log-phase HEK 293 cells on microscope PLA signal was detected for NEIL1–PCNA interaction in S-phase coverslips were pulse-labeled with BrdU (10 min); the cells were fixed, stained cells, and NEIL2–PCNA association was markedly higher in with Abs for BrdU (Abnova) and NEIL1/2 (11, 21) or PCNA (Santa Cruz) and NEIL1-depleted cells (Fig. 5C). These results provide additional distinct fluorescence-tagged secondary Abs. PCNA was used as a positive con- support for our conclusion that NEIL1 is preferentially associated trol. Images were captured by using a Carl Zeiss LSM 510 confocal microscope. with replication proteins in the S phase, and that NEIL2 serves as a backup for NEIL1 in repairing the replicating genome. the S-phase chromatin (Fig. 3B), whereas NEIL2 level colo- NEIL1 Coopts DNA Replication Proteins to Carry Out Repair of calized with RF-C in G - or S-phase cells was presumably too low 1 Oxidized Bases. During BER, the DNA glycosylase sequentially to be detected under our condition of quantitative PCR. The excises the base lesion and cleaves the DNA strand at the damage difference in the level of immunoprecipitated DNA was further ′ fi site via its intrinsic lyase activity to generate a 3 -blocking phospho- con rmed by comparison of cycle threshold (Ct) values after real- α,β-unsaturated aldehyde or phosphate moiety. In mammalian time PCR analysis of the products (plotted as fold enhancement cells, these groups are then removed by APE1 or PNKP, respec- compared with IgG in Fig. 3C). Taken together, these results tively, followed by the filling of the 1-nt gap by Polβ and nick show that NEIL1 but not NEIL2 colocalized with the DNA rep- sealing by LigIII during single-nucleotide incorporation or short- lication complex in S-phase cells. patch base-excision repair (BER) (1, 26). In contrast, the long- patch BER (LP-BER) subpathway, initially identified in vitro, NEIL Immunocomplexes Contain DNA Replication Proteins. To char- involves repair synthesis of a longer (2–8 nt) segment by Polδ/e acterize the association of NEILs with the DNA replication pro- after downstream strand displacement (27–29). Participation of teins that may also be involved in BER, we tested for their DNA replication proteins in LP-BER includes FEN-1 to cleave presence in NEIL immunoprecipitates isolated from (RNase + the displaced flap, PCNA clamp and clamp loader RF-C, and LigI DNase)-treated nuclear extracts of HEK 293T cell lines stably for final nick sealing, suggesting that this repair process is acti- † transfected with NEIL1-FLAG, NEIL2-FLAG, or empty FLAG vated during DNA replication (30). We previously showed that expression plasmids. The levels of ectopic NEILs in these lines NEIL1 (and NEIL2) complexes with PNKP, Polβ, LigIIIα, and were comparable to those of the corresponding endogenous pro- fi δ XRCC1 are able to carry out ef cient single-nucleotide BER teins. The presence of Pol , RF-C, and LigI, in addition to PCNA when repair is reconstituted with these proteins in vitro (26, 31). and FEN-1, was observed in NEIL1-FLAG and NEIL2-FLAG IPs Similarly, NEIL1’s stable complexes with replication proteins but not in the vector control IP (Fig. 4A). Furthermore, after suggested that it also can carry out LP-BER by using the repli- normalizing to the FLAG level, the amounts of PCNA, Polδ,RF-C, β β cation proteins. To test this, we eluted the proteins in NEIL1- FEN-1 and LigI, but not of DNA polymerase (Pol ), were FLAG IP from HEK 293 cells with a FLAG peptide (Sigma). found to be three- to sixfold lower in the NEIL2-FLAG IP rel- After confirming the presence of replication proteins in the eluate ative to the NEIL1-FLAG IP (Fig. 4A, Right). We also examined by Western analysis (Fig. 3A), we monitored the repair of a cir- the complexes of endogenous NEILs with replication proteins by cular plasmid substrate (pUC19CPD) containing a single 5-OHU using NEIL1/NEIL2 Abs conjugated to IgA agarose beads lesion within a 32-nt sequence that could be isolated by digestion (Sigma). The NEIL1 and NEIL2 IPs from (DNase + RNase)- with Nt.BstNBI, an ssDNA-specific restriction endonuclease δ treated HEK 293 cell extracts contained Pol , RF-C, and FEN-1 (Fig. 6A) (32). To distinguish between single-nucleotide and (Fig. 4B), which were absent in the control IP with rabbit IgG. The in-cell physical association of endogenous NEIL1 with fi † DNA replication proteins was further con rmed by in situ Mitra S, et al., Proceedings of Princess Takamatsu Symposium, November 10–12, 2009, Proximity Ligation Assay (PLA; Duolink kit; cat. no. LNK- Tokyo, Japan.

E3092 | www.pnas.org/cgi/doi/10.1073/pnas.1304231110 Hegde et al. Downloaded by guest on October 1, 2021 PNAS PLUS BIOCHEMISTRY

Fig. 3. Enrichment of NEIL1 and DNA replication proteins in the same genomic sequences of S-phase cells. (A) ChIP/re-ChIP assay: NEIL1 and replication proteins

have a stronger association with DNA in S-phase cells than in G1 cells. ChIP assay with IgG or NEIL1 Ab (first IP) of cross-linked chromatin from HEK 293 cells at G1 vs. S phase, PCR amplification products of a 250-bp region in the GAPDH or β-actin gene (primer sequences in Fig. S1B). Second IP of other four fractions with IgG (control) or Ab for replication proteins and PCR amplification as before. (B) Reverse ChIP was performed with the first IP by using RF-C Ab and the second IP with NEIL1, NEIL2, or PCNA Abs. (C) ChIP products were quantified by real-time PCR, and Ct values are plotted as fold enhancement after normalizing to IgG. (D) Cell-cycle distribution

of G1 vs. S phase cells showed ∼55% cells in S phase 3 h after release from double thymidine block. Other details are provided in Materials and Methods.

LP-BER, the repair patch size was monitored by using various level, we investigated NEIL2’s ability to carry out LP-BER by 32P-labeled dNTPs in the reaction (33). Incorporation of using these proteins as before. Because lesion base excision is the 32P-labeled dNMPs by NEIL1 IP indicated a LP-BER patch size rate-limiting step in overall repair, we first normalized the base of at least 2, 3, and 4 nucleotides, respectively (Fig. 6B). The excision activity of NEIL1 vs. NEIL2 by adjusting their levels in presence of a small amount of unligated intermediates suggests that eluates from NEIL-FLAG IPs from HEK 293 cells (Fig. 7A)to the DNA ligase was limiting in the IP eluate. These results showed achieve comparable 5-OHU excision levels (Fig. 7B). It was ob- that the protein complex eluted from NEIL1 IP from human cells served that 0.1 and 0.2 μg of NEIL1 IP had DNA glycosylase contains all the essential components to proficiently carry out repair activity comparable to 0.4 and 0.8 μg of NEIL2 IP, respectively. of 5-OHU lesion via LP-BER subpathway. The dependence of As denoted by the dotted line in the histogram in Fig. 7B NEIL1 IP-initiated LP-BER on replication proteins was fur- (Lower). The NEIL1 IP produced approximately four to fivefold ther confirmed by the lack of enhanced repair by addition of higher level of repair than the NEIL2 IP with comparable repair- recombinant Polβ;incontrast,Polδ significantly stimulated the initiating glycosylase activity (Fig. 7C). repair (Fig. 6C). Similar studies that used reconstituted systems with purified DNA replication proteins (indicated in Fig. S2) and containing Collaboration of DNA Replication Proteins with NEIL1 vs. NEIL2 for comparable activities of NEIL1 and NEIL2 (Fig. S3A) confirmed Oxidized Base Repair. In view of our finding that NEIL2 associates approximately fourfold higher repair with NEIL1 that with NEIL2 with DNA replication proteins like NEIL1, albeit at a much lower (Fig. S3B). Taken together, these results underscored preferential

Hegde et al. PNAS | Published online July 29, 2013 | E3093 Downloaded by guest on October 1, 2021 Fig. 4. In-cell association of NEIL1 and NEIL2 with DNA replication proteins. (A) FLAG IPs from DNase I/RNaseA-treated (100 μg/mL each) extracts of HEK 293 cells ectopically expressing empty or NEIL1- or NEIL2-FLAG (1, 21) were analyzed for replication and other repair proteins. Quantitation of immunoblot bands (histogram; Right) shows approximately three- to fivefold higher levels of replication proteins (Polδ, PCNA, RF-C, FEN-1, LigI) in the NEIL1 IP than in the NEIL2 IP, whereas the Polβ levels in these IPs are similar. (B) Endogenous IP of HEK 293 cell extracts with NEIL1 or NEIL2 Ab or IgG bound to protein A beads confirmed stable association of NEILs with Polδ, RF-C, and FEN-1. (C) In situ PLA assay (Duolink) demonstrating the association of NEIL1 with Polδ, RF-C, FEN-1, and LigI in HEK 293 cells. PLA with NEIL1 Ab (rabbit) vs. IgG (mouse) served as a control.

association of NEIL1 with the replication proteins to carry out Similar analyses with other DNA glycosylases NEIL2 and prereplicative LP-BER of oxidized bases, and also suggested that NTH1 showed that DNA synthesis by Polδ was prevented by NEIL2 could serve as a backup glycosylase in NEIL1’s absence. NEIL2 but not NTH1 (Fig. S4A). Furthermore, whereas NEIL2’s binding, like that of NEIL1, was higher for 5-OHU–containing NEIL1 Blocks DNA Synthesis by Polδ in a Template Containing 5-OHU RPA-coated partial duplex compared with the control oligo, Lesion. To gain further insight into how NEIL1 coordinates the NTH1’s binding to the two oligos were comparable (Fig. S4B). repair of oxidized bases at the replication fork, we tested the These results further support NEIL2’s backup function for effect of NEIL1 on DNA synthesis by Polδ with a replication NEIL1 when needed. The lack of Polδ stalling by NTH1 (which fork-mimicking RPA-coated primer-template substrate contain- does not bind to base lesions in ssDNA) was expected, so NTH1 ing 5-OHU in the template (Fig. 8A). Incorporation of [32P] served as a control. dCMP (together with unlabeled dNMPs) indicated that DNA synthesis past the 5-OHU lesion occurred in the absence of Discussion NEIL1 (Fig. 8A, lanes 2–3). Excess PCNA in the reaction The ssDNA template at the replication fork may be more prone (fivefold molar excess) significantly enhanced Polδ-mediated to oxidative base damage and strand breaks than nonreplicating DNA synthesis, as expected (Fig. 8A, lane 3). Presumably, the DNA, thus warranting its urgent repair to prevent mutations (4). PCNA clamp loading at the end, slides on and off the duplex Repair of mutagenic bases incorporated during replication [i.e., postreplicative repair (34, 35)] is also essential for maintaining region of the substrate, so that equilibrium is maintained be- fi tween bound and free PCNA. The presence of NEIL1, whose genomic delity. 8-Oxoguanine (8-oxoG), a predominant ROS- strand-cleavage activity is prevented by RPA (Fig. 8B) (13), induced base lesion in DNA, could be mutagenic if not repaired before replication, because replicative DNA polymerases (i.e., blocked DNA synthesis by Polδ past the lesion site even in the Polδ/e) often incorporate A opposite 8-oxoG in the template strand, presence of PCNA (Fig. 8A, lanes 4–5). The lack of strand generating an A:8-oxoG mispair (36). However, this mismatch is cleavage by NEIL1 under the optimal repair conditions was fi fi 32 ′ ef ciently repaired by MYH (35), a mammalian homologue of con rmed by carrying out a similar reaction containing a P-5 - Escherichia coli MutY that excises A from the 8-oxoG:A (and end labeled template strand and unlabeled dNTPs (in the pres- δ possibly FapyG:A) pairs (37). Similarly, the U:A pair generated ence of RPA, Pol , and PCNA as before; Fig. 8B). These data as a result of incorporation of U during replication is repaired strongly support a scenario whereby NEIL1 nonproductively postreplicatively by uracil-DNA glycosylase (UNG2) (34, 38). binds to the 5-OHU lesion in the RPA-coated partial duplex Like NEIL1, UNG2 and MYH associate with PCNA at the δ oligo and blocks nascent chain elongation by Pol . To further replication foci (39, 40), which presumably recruit these at the confirm this, we performed affinity coelution of NEIL1 with replication sites, facilitating postreplicative repair of inappropriate RPA-coated partial duplex oligo with or without 5-OHU located bases (40). However, whether these glycosylases are coupled to in the ss segment, and used streptavidin pull-down analysis (Fig. the replication machinery or they bind to PCNA molecules at the 8C). Consistent with its stalling of Polδ, NEIL1’s binding was newly replicated DNA is not clear (35). In contrast, removal of U, markedly higher when the oligo contained 5-OHU. These data generated by deamination of cytosine in DNA, needs to be clearly suggest that NEIL1’s high-affinity binding to base lesions prereplicatively repaired. Similarly, repair of most other oxidized in an ssDNA template (8) stalls progression of the replication base lesions (e.g., 5-OHU, glycol, 5-OHC, FapyA, 8-oxoA, fork, thus allowing prereplicative repair. uracil glycol) in the template DNA, which are primary NEIL

E3094 | www.pnas.org/cgi/doi/10.1073/pnas.1304231110 Hegde et al. Downloaded by guest on October 1, 2021 of DNA synthesis by Polδ at the lesion site in the template is PNAS PLUS presumably required for prereplicative repair. Furthermore, NEIL1’s localization in replicating DNA foci and enhanced as- sociation with replication proteins in S-phase cells, resulting in its enhanced activity, indicates its ability to coopt replication pro- teins to repair base lesions at the replication fork. We propose that NEIL1 is a component of the DNA repli- cation complex needed for surveillance of oxidized bases before replication, and thus acts as a “cowcatcher” (Fig. 9). Its prefer- ence for binding to lesions in ssDNA (8) should help target such lesions at the replication fork. RPA, which coats the ssDNA at the replication fork, inhibits NEIL1’s strand scission activity via direct interaction, presumably to prevent double-strand break formation (Fig. 9) (13), where repair is not possible because of the lack of the complementary strand. We postulate that NEIL1’s stalling of the replication complex (Fig. 8) causes regression of the replication fork to form a “chicken-foot” structure. The reannealing of the template strand restores the lesion into the duplex region, where its repair is initiated with its excision by NEIL1 followed by repair synthesis by the replication proteins (42). Replication resumes after resolution of the stalled fork. The helicase activity of WRN, which functionally interacts with NEIL1 (9) and other replication proteins, including PCNA (43), RPA (44), and FEN-1 (45, 46), has been implicated in replica- tion fork regression and resolution (44, 47, 48). Alternatively, Smarcal1 (and other annealing helicases) could also be involved in resolving the chicken-foot structure (49, 50), although its in- teraction with NEIL1 has not yet been tested. We observed inhibition of chain elongation after oxidative stress in NEIL1-depleted cells (Fig. 1). Although this result appears to be counterintuitive, there are two possible explanations. First, it is likely that, even when NEIL1 bound to replicating DNA at the lesion site causes replication arrest followed by fork collapse, re- gression, lesion repair, and replication restart, the delay in repli- cation is too short to be detected in the fiber growth assay. On the contrary, when NEIL2 takes over in the absence of NEIL1, and BIOCHEMISTRY carries out the same steps but at a much slower rate, the resulting delay is large and is reflected in slowdown of the fiber growth. An alternative possibility is that thymine glycol, which blocks DNA replication, is efficiently repaired by NEIL1. In NEIL1-depleted cells, inefficient repair of this lesion by alternative means was reflected in slower nascent chain growth. We favor the first Fig. 5. Enhanced association of NEIL1 with PCNA in S-phase cells and of possibility because, in the absence of NEIL1, NEIL2 acts in a NEIL2 with PCNA in NEIL1-depleted cells. (A and B) Endogenous PCNA was manner qualitatively similar to NEIL1 (Fig. S4). However, its immunoprecipitated from extracts of HEK 293 cells in G1 vs. S phase with repair efficiency is significantly lower than that of NEIL1 (Fig. 7), PCNA Ab (Santa Cruz). (A)Western analysis with NEIL1 and NEIL2 Abs which could be partly caused by a lack of its activation by repli- showed a strong association only of NEIL1 with PCNA in S phase vs. G1 cells. (B) Enhanced association of NEIL2 with PCNA in NEIL1-depleted cells. (C) cation proteins (10). We previously showed that NEIL2 is pref- (Upper) In situ PLA assays showing NEIL1’s strong interaction with PCNA in erentially involved in repair during transcription (21). Taken S-phase cells and NEIL2’s enhanced interaction in NEIL1-depleted HEK 293 together, these data suggest that, in the absence of NEIL1, NEIL2 cells. (Lower) PLA signals for NEILs and PCNA in IgG controls. acts as a “relief pitcher” in removing cytotoxic base lesions from the replicating genome. We should point out in this context that recently characterized substrates, could not be repaired postreplicatively in an error- NEIL3 was also shown to be activated during the S-phase (51). free manner, and hence must be repaired before replication to However, human NEIL3’s substrate preference has not been prevent . The 5-OHU lesion is potentially the most well characterized. It is possible that NEIL3 is also involved in mutagenic among these (3). However, how prereplicative repair prereplicative repair for a different set of oxidized bases. On the could be carried out in the template DNA without causing contrary, although not an oxidized base, U generated by oxida- double-strand breaks has not been elucidated. tive deamination of C in the template DNA also needs to be Our cumulative observations helped to identify unique fea- repaired prereplicatively. Based on the studies of Krokan et al., it tures that set the NEILs apart from OGG1 and NTH1, including appears that nuclear UNG2 may function in an analogous the NEILs’ ability to recognize ss DNA substrates (8). Further- fashion as NEIL1 and that SMUG1 could serve as the backup more, NEIL1 is active with base lesions (e.g., 8-oxoG or 5-OHU) enzyme for U repair (34, 38, 40). in the primer strand recessed from the 3′-terminus by 1 to 3 nt, Together, these observations led us to propose that the broad whereas OGG1 and NTH1 are inactive with such substrates (41). and overlapping substrate specificity of oxidized base-specific In this report, we have provided direct evidence for NEIL1’s glycosylases ensures their ability to provide a backup function † preferential role in repairing oxidized bases in coordination with when needed (1, 40, 52). This is consistent with the observation DNA replication proteins. NEIL1 is thus required for replication that mouse mutants lacking individual glycosylases, and cells de- fork progression in oxidatively stressed cells. The observed stalling rived therefrom, are viable, without strong phenotypes (53–55).

Hegde et al. PNAS | Published online July 29, 2013 | E3095 Downloaded by guest on October 1, 2021 Fig. 6. NEIL1 coopts replication proteins to carry out LP-BER. (A) The plasmid substrate for our BER assay is shown: a 32-nt NtBstNB1 (57) restriction fragment with a 5-OHU at residue 20 is monitored for repair. Incorporation of labeled bases downstream from 5-OHU indicates LP-BER indicates LP-BER. (B) FLAG-NEIL1 IP from HEK 293 cells carries out LP-BER of 5-OHU. Total proteins eluted with FLAG peptide (1, 5, and 10 μg) were used for incorporation of [α-32P]dAMP (lanes 2–4), dGMP (lanes 5–7), or dCMP (lanes 8–10). After digesting the plasmid with Nt.BstNB1, the 32-nt repaired fragment was analyzed by denaturing elec- trophoresis (32). Lane 1 shows repair using empty FLAG vector IP. The 32P-5′-end labeled 32 and 20 nt oligos were used as size markers (lane 11). (C) Addition of recombinant Polδ but not Polβ (50 and 100 fmol) to NEIL1 IP (1 μg) enhanced LP-BER (second nucleotide incorporation). Other details are provided in Materials and Methods.

In any case, the present study documents a mechanism of pre- Cell Culture and Coimmunoprecipitation. The human embryonic kidney cell replicative repair of oxidized DNA bases in mammalian genomes line HEK 293, grown at 37 °C and 5% CO2 in DMEM containing 10% FBS and in which the repair enzyme acts as a component of the rep- 100 U/mL each of penicillin and streptomycin, was transfected with empty or NEIL1- or NEIL2-FLAG–expressing plasmids. We also generated FLAG-NEIL1 lication complex. or NEIL2-expressing stable HEK 293 and human colorectal tumor line, HCT116, using Zeocin as the selection marker as described previously (31). At 48 to Materials and Methods 56 h after transient transfection, or at ∼80% confluence in the case of stably Preparation of Single-Lesion, 5-OHU–Containing Oligonucleotide and Plasmid expressing cultures, the cells were harvested, lysed, and treated with 500 Substrates. A 51-mer oligo containing 5-OHU at position 26 from the 5′ end, U/mL each of DNase I and RNase A (Ambion) at 37 °C for 30 min, cleared by and undamaged complementary oligos, containing G opposite the lesion, or centrifugation, and immunoprecipitated by rocking for 3 h at 4 °C with FLAG sequences for producing oligos for ligase and polymerase assay (Fig. S1), M2 Ab crosslinked to agarose beads (Sigma) as described before (11, 13). were purchased from Midlands. The 32P-5′-end labeling of the oligos, anneal- Similarly, endogenous NEILs were immunoprecipitated using anti-NEIL1 or NEIL2 Abs conjugated to IgA agarose beads. The beads were collected by ing, and purification of labeled oligos were described previously (11). Circular centrifugation, washed three times with cold TBS plus 0.1% Triton X-100, plasmid substrate, pUC19CPD containing a single oxidized base lesion, 5-OHU, and FLAG-NEILs were eluted from the beads. After elution from the beads was generated as described previously (32, 56, 57). Covalently closed form I by adding SDS loading buffer, the immunocomplex was separated in 12% fi plasmid was puri ed by CsCl centrifugation, and the presence of the 5-OHU SDS/PAGE and immunoblotted by using Abs for Polδ, RF-C, LigI, Polβ, LigIIIα, fi lesion was veri ed by agarose gel electrophoresis after treatment with NEIL1, FEN-1 (Bethyl Laboratories), and the appropriate secondary Abs. which converted the plasmid to a nicked circle (form II). Cell Synchronization and Cell Cycle Analysis. HEK 293 cells were synchronized Expression and Purification of Recombinant Proteins. Recombinant WT NEIL1, by double thymidine block as described earlier (61). Briefly, at ∼40% con- NEIL2, PNKP, PCNA, Polδ, RF-C, DNA LigI, FEN-1, Polβ, and LigIIIα were pu- fluence, cells were treated with 10 mM thymidine for 18 h, released for 4 h rified as described previously (5, 6, 10, 11, 58–60). by adding fresh media after washing with PBS solution, and subjected to

Fig. 7. NEIL1 is more efficient in LP-BER than NEIL2 when normalized to the same specific glycosylase activity. (A) FLAG IPs of FLAG-tagged NEIL1 and NEIL2 were adjusted for equal 5-OHU excision activity, based on the amount of protein by Western blotting with anti-FLAG Ab (B) and glycosylase activity (dotted line in histogram) using a 5-OHU–containing 32P-5′-end labeled duplex oligo substrate. (C) LP-BER assay using a 5-OHU–containing plasmid (Fig. 6) shows approximately fivefold higher repair with NEIL1 IP compared with NEIL2 IP.

E3096 | www.pnas.org/cgi/doi/10.1073/pnas.1304231110 Hegde et al. Downloaded by guest on October 1, 2021 PNAS PLUS

Fig. 8. NEIL1’s nonproductive binding to the base lesion 5-OHU in a partial duplex oligo stalls DNA synthesis by Polδ.(A) A partial duplex, as indicated, represents a primer template for DNA replication with Polδ in which the ss segment (29 nt) in the template, with 5-OHU at residue 27, is complexed with RPA. The reaction contained 5 nM substrate, 10 nM RPA, 5 nM each of Polδ and PCNA, and 10 nM of NEIL1. Addition of excess PCNA (DNA:PCNA ratio, 1:5) enhances polymerase activity as monitored by [32P]dCMP incorporation at residue 29 (lane 3). DNA synthesis was prevented by NEIL1 (lane 5). (B) RPA inhibits NEIL1’s base excision/abasic site (AP) lyase activity. The RPA bound to the template strand (RPA:DNA molar ratio, 1:3) prevents NEIL1-catalyzed cleavage ofthe 32P-5′-end-labeled template strand at the 5-OHU site under the same condition as in A.(C)Affinity coelution of NEIL1 with 5′ biotinylated partial duplex oligo (with or without 5-OHU) bound with streptavidin-agarose beads (Invitrogen). The ss segments of the oligo were coated with RPA as in A. NEIL1 binding was significantly higher when the oligo contained 5-OHU compared with the oligo without the lesion.

a second thymidine treatment (10 mM) for 17 h. Cells were then stimulated for 30 min. An equal volume of high-salt buffer (3% polyethylene glycol, to proliferate with fresh media and harvested at 0, 2, 3, 4, 5, 6, and 8 h and 5 μg/mL propidium iodide, 0.1% Triton X-100, and 400 mM NaCl) was added, processed for cell cycle analysis (62). Cells were suspended in a low-salt and the cells were kept at 4 °C overnight. The cellular DNA content was buffer containing 3% polyethylene glycol, 5 μg/mL propidium iodide, 0.1% evaluated by flow cytometry by using a FACScan flow cytometer (Becton Triton X-100, 4 mM Na citrate, and 100 μg/mL RNase, and incubated at 37 °C Dickinson). Histograms were analyzed by using ModFit LT cell cycle analysis software (Verity Software House) to determine the percent of cells in various stages of the cell cycle. A total of 10,000 events were collected for all samples.

DNA Fiber Analysis. To measure replication rates in HEK 293 cells after NEIL1 and/or NEIL2 depletion, cells transfected with specific siRNA for NEIL1 (se- quence: sense, 5′CCGUGAUGAUGUUUGUUUAUU3′;antisense,5′UAAA- CAAACAUCAUCACGGUU3′; Sigma) or NEIL2 (25) for 48 h were sequentially

treated with CldU (C6891; 20 μM/20 min; Sigma), H2O2 (1 mM, 10 min), and IdU (I7125; 50 μM, 20 min; Sigma) (14, 15). Cells were rinsed with PBS solu- tion three times after each treatment. Cells were then harvested by scraping BIOCHEMISTRY in cold PBS solution and diluted to a concentration of 5 × 105 cells per milliliter. A drop of cell suspension together with spreading buffer (200 mM Tris·HCl, pH 7.4, 50 mM EDTA, and 0.5% SDS) was put on a microscope slide and incubated for a few minutes to allow lysis. The slide was then tilted so that DNA fibers could spread over it. Slides were air-dried and the fibers fixed on the slides with methanol/acidic acid (3:1). Fibers were stained with monoclonal rat anti-BrdU [clone BU1/75 (ICR1); Oxford Biotechnologies] for CldU and monoclonal mouse anti-BrdU (clone B44; no. 347580; Becton Dickinson) for IdU. Secondary Abs were goat anti-rat Alexa Fluor 555 and goat anti-mouse Alexa Fluor 488. DNA fibers were visualized by fluorescence microscopy. The lengths of CldU and IdU tracks were measured by using ImageJ software, and micrometer values were converted into kilobases by using Alkylation-Induced Replication Block 79 conversion factor (1 μm = 2.59 kb). At least 100 forks were analyzed for every condition.

ChIP/Re-ChIP Assay. ChIP analysis was performed with HEK 293 cells by using a ChIP assay kit (Cell Signaling Solution; Millipore) per the manufacturer’s protocol, and the re-ChIP assays were performed as described previously (21, 63).

In Situ PLA. HEK 293 cells grown overnight in 16-well chamber slides were fixed with 4% paraformaldehyde, then permeabilized with 0.2% Tween 20, followed by incubation with a primary Ab for NEIL1 (rabbit) (5) or DNA replication proteins (mouse monoclonal, as indicated). The PLA assay was performed using the Duolink PLA kit (cat. no. LNK-92101-KI01; OLink Bio- science) per the manufacturer’s instructions. The nuclei were counterstained with DAPI, and the PLA signals visualized in a fluorescence microscope (Olympus) at 200× magnification (56, 64).

Fig. 9. Cowcatcher model for prereplicative oxidized base repair by NEIL1. DNA Glycosylase Assay. DNA strand scission catalyzed by NEIL1 or NEIL2 was We propose that NEIL1 acts as a cowcatcher in the replication complex when analyzed with 32P-5′-end labeled 51-mer oligo substrates containing 5-OHU it stalls progression of the replicative polymerase at the lesion site, leading at position 26 from the 5′ end (11) (Fig. S1). A total of 2 pmol of substrate to fork regression, as a result of which the damage in the reannealed duplex was incubated with NEIL1 or NEIL2 (indicated amounts) or HEK 293 cell can be repaired by NEIL1-initiated BER using the replication proteins. WRN nuclear extracts at 37 °C for 15 min in a 10-μL reaction mixture containing helicase, which interacts with NEIL1 (9), and other replication proteins (43– 40 mM Hepes, pH 7.5, 50 mM KCl, 100 μg/mL BSA, and 5% glycerol. After the 45) resolve the chicken-foot after completion of repair (47, 48). reaction was stopped with formamide dye (80% formamide, 20 mM NaOH,

Hegde et al. PNAS | Published online July 29, 2013 | E3097 Downloaded by guest on October 1, 2021 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol), the DNA (pUC19CPD) or duplex oligo with blocked (biotinylated) ends (Fig. S1)in products were separated on a 20% polyacrylamide gel containing 8 M urea in the presence of dNTPs plus one radiolabeled [α-32P]dNTP as indicated. The 1× Tris/borate-EDTA buffer, pH 8.4 (11, 58). The radioactivity was quantified by 20-μL reaction mixture contained 200 fmol of damage-containing plasmid using a PhosphorImager (Amersham Biosciences) and Image Quant software. substrate, 1 mmol ATP, 25 μmol unlabeled dNTPs, and 10 μmol [α-32P]dNTPs (the concentration of the corresponding cold dNTP was lowered to 5 μM, Affinity Coelution of Biotinylated DNA Oligo. A partially duplex oligo (with or unless otherwise specified) in BER buffer (25 mM Hepes·KOH, pH 7.9; 50 mM ′ without 5-OHU within the ss segment) with a biotin residue at the 5 end of KCl; 2 mM MgCl2; 0.5 mM DTT). After incubation for 30 min at 37 °C, the the longer strand was mixed with RPA (DNA:RPA at 1:2 molar ratio). The plasmid DNA was phenol/chloroform-extracted, ethanol-precipitated, recovered, oligo was then bound to magnetic streptavidin beads (Invitrogen). After and digested with N.BstNBI (New England Biolabs), then resolved on a de- extensive washing with TBS buffer, the beads were mixed with NEIL1, NEIL2, naturing polyacrylamide gel and analyzed with a PhosphorImager. or NTH1 (1:2 molar ratio to DNA) for 1 h with constant rotating. The bound fi proteins were eluted with SDS buffer and identi ed by Western analysis. ACKNOWLEDGMENTS. We thank Priscilla Cooper and Miaw-Sheue Tsai (Lawrence Berkeley National Laboratory) for baculovirus expression of RF-C Reconstitution of NEIL-Initiated LP-BER. For repair assays that used immuno- and Polδ in the Expression and Molecular Biology Core (P01 CA92584); for- complexes isolated from empty FLAG-, NEIL1-FLAG- or NEIL2-FLAG–expressing mer S.M. laboratory members A. Das and C. Theriot for various discussions cells (transiently or stably), the immunocomplexes were eluted from FLAG- and David Konkel for editing the manuscript; and the anonymous reviewers of the original manuscript for suggesting studies that enhanced the quality agarose beads with FLAG peptide (1×; Sigma) at 4 °C after constant rota- of the revised version. This work was supported by US Public Health Service tional mixing, in a buffer containing PBS solution, 1 mM DTT, and 10% Grants R01 CA81063 (to S.M.), P01 CA092584 (to A.E.T. and S.M.), R01 glycerol. An aliquot of the eluate was tested for the presence of DNA repli- GM57479 (to A.E.T.), R01 NS073976 (to T.K.H.), R01 ES018948 (to I.B.), and cation proteins by immunoblotting. The proteins eluted from NEIL immu- R01 CA167181 (to G.-M.L.); and Alzheimer’s Association New Investigator nocomplexes were used in repair reactions with the 5-OHU–containing plasmid Research Grant NIRG-12-242135 (to M.L.H.).

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