The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD - PubMed (original) (raw)
The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD
Pavana M Hegde et al. J Biol Chem. 2015.
Abstract
The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.
Keywords: BERosome; DNA damage; DNA damage response; DNA polymerase; DNA replication; NEIL1 DNA glycosylase; base excision repair (BER); dominant negative inhibition; prereplicative repair.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Figures
FIGURE 1.
NEIL1 forms a specific BER-proficient multiprotein complex with DNA replication proteins in human cells that contains PNKP but not APE1. A, HEK293 cell nuclear extracts were precleared with benzonase and fractionated on a Sephacryl 300 gel filtration column. A significant fraction of NEIL1 elutes as megadalton-size complex (>2000 kDa), which contains DNA replication proteins. B, complete LP-BER assay was performed with 5′-biotinylated duplex oligonucleotide substrate (51 nucleotides long) with 5-OHU at position 26. Sephacryl fractions 44 and 45, containing NEIL1 and replication proteins, showed repair activity. In contrast, fraction 80, mostly containing uncomplexed NEIL1 and a small amount of PNKP and Polβ, generated unligated repair products. Complete BER was achieved with fraction 62 when supplemented with recombinant PNKP, Polβ, and DNA ligase IIIα (_LigIII_α). C and D, co-IP of FLAG-NEIL1 from HEK293 cells contained PNKP but not APE1; conversely, co-IP of FLAG-OGG1 contained APE1 but not PNKP, suggesting that NEIL1 and OGG1 form unique BER complexes containing essential downstream repair proteins. E–G, CRISPR/Cas-mediated NEIL1 KO in HEK293 cells (E) does not affect RFC in cellulo association with PCNA, Polδ, or LigI, as analyzed by RFC co-IP (F) and PLA (G).
FIGURE 2.
Pairwise interaction of NEIL1 with DNA replication proteins in vitro via CTD. A, interaction with RFC. a, in vitro His tag affinity analysis with recombinant proteins shows that both NEIL1 and NEIL2 directly interact with His-RFC. b, GST-NEIL1 CTD fragments with aa 312–389 and 312–349 but not aa 350–389 co-elute with RFC. GST alone was used as the control. B, interaction with Polδ. a, full-length NEIL1 and its CTD peptide aa 289–389 but not aa 1–288 polypeptide were co-eluted with His-Polδ. WT NEIL2 also co-eluted with His-Polδ. b, as with RFC, Polδ was co-eluted with GST-NEIL1 CTD peptides aa 312–389 and 312–349 but not with aa 350–389. c, Far-Western analysis of Polδ shows its interaction with NEIL1(WT) and the polypeptide aa 1–349 but not the aa 1–311 peptide. FEN1 served as a positive control. d, reverse Far-Western analysis (Polδ on the membrane with four subunits) shows interaction of NEIL1 with p66 and p50 subunits. Polβ served as the positive control. C, interaction with LigI. a, Far-Western analysis of NEIL1 CTD peptides indicates that LigI binding requires aa 289–349 in the NEIL1 CTD. b, co-elution of LigI with GST-NEIL1 fragments aa 289–389 and 289–349 but not aa 312–389 or 312–349. D, the interaction mapping of NEIL1 domains with RFC, Polδ, and LigI shows distinct but overlapping binding residues within the CTD.
FIGURE 3.
Loss of CTD abrogates in cellulo association of NEIL1 with DNA replication proteins. A, FLAG-IP from FLAG-NEIL1(N311)-expressing HEK293 cells shows a significantly reduced level of LigI, RFC, and Polδ, but not PCNA, compared with that from FLAG-NEIL1(WT)-expressing cells. PCNA binding requires residues 289–312, as shown previously (11). B, PLA of WT versus N311 mutant NEIL1-FLAG with replication proteins confirms that CTD is required for binding. C, quantitation of PLA foci from 25 or more cells. Error bars, S.E.
FIGURE 4.
Activation of NEIL1 but not NEIL2 by RFC via pairwise interaction. A, 5-OHU lesion excision/lyase activity of NEILs with 5′-32P-labeled primer-template oligonucleotide substrate shows that RFC stimulates WT NEIL1 by ∼8-fold but does not affect N311 mutant lacking the CTD. NEIL2 activity was also not affected. B, RFC stimulated NEIL1 activity with duplex oligonucleotide substrate, albeit to a lesser extent compared with the primer-template substrate. C, kinetic parameters reveal RFC stimulation of NEIL1 activity primarily by increasing its turnover (∼5-fold increase in _K_cat), although a ∼2.5-fold decrease in Km suggested an increase in substrate affinity as well. D–E, RFC activates NEIL1-initiated LP-BER (lanes 5 and 6) with DNA replication proteins. Repair was carried out with pUC19CPD plasmid substrate containing a single 5-OHU lesion (D) (5, 14). The LP-BER reaction was optimized for linear dose dependence for the NEIL1 level (lanes 7 and 8). Error bars, S.E.
FIGURE 5.
Pairwise interaction and differential stimulation of NEILs with FEN-1. A, Far-Western analysis and His tag co-elution of NEIL1 and NEIL2 with WT FEN1 and its C-terminal domain (aa 328–380). B, BER activity with 5-OHU-containing bubble oligonucleotide (33) shows that FEN-1 stimulates NEIL1 but not NEIL2. C and D, binding (Kd) measurement using fluorescence spectroscopy (12, 14) showed 3-fold reduced affinity of the FEN-1 CTD (aa 328–380) for NEIL2 relative to NEIL1. The FEN-1 CTD lacking Trp or Tyr residues has a negligible contribution to the fluorescence. Error bars, S.E.
FIGURE 6.
Loss of CTD abolishes recruitment of NEIL1 to chromatin and replication fork. A, immunoblotting of whole cell extracts, soluble nuclear fraction, and chromatin fraction from HEK293 cells transiently expressing FLAG-NEIL1(WT) or FLAG-N311 shows an absence of the latter in chromatin. B, immunofluorescence staining with FITC-conjugated anti-FLAG antibody shows comparable nuclear localization of N311 and WT NEIL1. C, real-time ChIP-PCR analysis at arbitrary genome sequences (RARβ2 or CDKN1A promoter regions) in double-thymidine block-synchronized HEK293 cells at the G1/S boundary versus S phase shows a requirement of CTD for S phase-specific recruitment of NEIL1 to chromatin. D, log phase HEK293 cells ectopically expressing WT NEIL1-FLAG or N311-FLAG were pulse-labeled with BrdU. PLA was performed with anti-FLAG and anti-BrdU Ab. A significant number of PLA foci in WT NEIL1-expressing cells indicates its recruitment at the replication fork/foci. An absence of PLA foci in N311-expressing cells confirms the requirement of the CTD. 25 cells were analyzed for quantitation. Error bars, S.E.
FIGURE 7.
The CTD is dispensable for NEIL1 base excision/AP lyase activity but is required for optimum repair activity involving replication proteins. A, complete LP-BER assay with pUC19CPD as in Fig. 4_D_, using recombinant proteins, shows proficient repair with WT NEIL1 but not with the nested CTD deletion polypeptides aa 1–349 and 1–311. The 1–288 mutant was inactive, as shown previously (30). B, similar results were obtained with linear duplex oligonucleotide substrate for WT NEIL1 and N311 mutant. C, base excision/AP lyase activity was comparable for WT NEIL1, 1–349, and 1–311. The 1–288 polypeptide was inactive. Error bars, S.E.
FIGURE 8.
Isolated CTD peptide of NEIL1 dominant negatively inhibits BER in vitro. A, 5-OHU-containing duplex oligonucleotide, end-biotinylated, used in the LP-BER assay. B, dose-dependent inhibition of LP-BER activity by CTD (aa 312–389; lanes 1–5). The non-interacting CTD (aa 350–389) peptide does not affect BER (lanes 6–8). LP-BER activity of FLAG-NEIL1 IP was inhibited by recombinant CTD peptide (lanes 9 and 10). AU, arbitrary units. Error bars, S.E.
FIGURE 9.
Ectopic introduction of CTD peptide sensitizes cells to oxidative stress by inhibiting BERosome formation. A, flow diagram depicting the protocol to measure genome damage/repair after transduction of TAT/GFP-fused CTD peptides or transfection of FLAG-CTD plasmid, which was confirmed by immunofluorescence or FITC-conjugated anti-FLAG antibody (B). C, estimation of oxidized base lesions by long amplicon PCR. Glucose oxidase (GO)-induced DNA damage (measured at 0.5 and 5 h after glucose oxidase treatment) in HEK293 cells was measured by long amplicon PCR of Fpg/EndoVIII-treated genomic DNA. Persistent damage was identified at 5 h in CTD fragment 289–389-expressing cells but not in 350–389-expressing cells. D, TAT-CTD peptide transduction reduced RFC association with PCNA, Polδ, or LigI as analyzed by PLA. Quantitation is shown as a histogram. E, a clonogenic cell survival assay shows reduced survival of glucose oxidase-treated HEK293 cells expressing FLAG-NEIL1(289–389) as compared with vector control or non-interacting peptide (aa 350–389). Error bars, S.E.
FIGURE 10.
Pairwise interaction between NEIL1, XRCC1, and Polβ stabilizes the ternary complex. A, analytical gel filtration chromatography profile of NEIL1 + XRCC1, Polβ + XRCC1, or NEIL1 + XRCC1 + Polβ. Whereas the two-protein mixtures formed only a negligible fraction of stable complex (peak 2), a significant portion of the mixture of three proteins exists in ternary complex (peak 1). B, Coomassie staining of the peak fractions confirms the ternary complex formation. Peaks 3, 4, and 5 represent monomeric XRCC1, NEIL1, and Polb, respectively.
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References
- Adhikari S., Choudhury S., Mitra P. S., Dubash J. J., Sajankila S. P., Roy R. (2008) Targeting base excision repair for chemosensitization. Anticancer Agents Med. Chem. 8, 351–357 - PubMed
- Begg A. C., Stewart F. A., Vens C. (2011) Strategies to improve radiotherapy with targeted drugs. Nat. Rev. Cancer 11, 239–253 - PubMed
- Bulgar A. D., Snell M., Donze J. R., Kirkland E. B., Li L., Yang S., Xu Y., Gerson S. L., Liu L. (2010) Targeting base excision repair suggests a new therapeutic strategy of fludarabine for the treatment of chronic lymphocytic leukemia. Leukemia 24, 1795–1799 - PubMed
- Hegde M. L., Hegde P. M., Bellot L. J., Mandal S. M., Hazra T. K., Li G. M., Boldogh I., Tomkinson A. E., Mitra S. (2013) Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins. Proc. Natl. Acad. Sci. U.S.A. 110, E3090–3099 - PMC - PubMed
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