Prereplicative repair of oxidized bases in the human genome 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 Cancer 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 mutations. It is unknown how endogenous and environmentally induced reactive oxygen species 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 enzyme. 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 base excision repair (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 gene 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).