title: NEIL1 responds and binds to psoralen-induced DNA interstrand crosslinks.
abstract: Recent evidence suggests a role for base excision repair (BER) proteins in the response to DNA interstrand crosslinks, which block replication and transcription, and lead to cell death and genetic instability. Employing fluorescently tagged fusion proteins and laser microirradiation coupled with confocal microscopy, we observed that the endonuclease VIII-like DNA glycosylase, NEIL1, accumulates at sites of oxidative DNA damage, as well as trioxsalen (psoralen)-induced DNA interstrand crosslinks, but not to angelicin monoadducts. While recruitment to the oxidative DNA lesions was abrogated by the anti-oxidant N-acetylcysteine, this treatment did not alter the accumulation of NEIL1 at sites of interstrand crosslinks, suggesting distinct recognition mechanisms. Consistent with this conclusion, recruitment of the NEIL1 population variants, G83D, C136R, and E181K, to oxidative DNA damage and psoralen-induced interstrand crosslinks was differentially affected by the mutation. NEIL1 recruitment to psoralen crosslinks was independent of the nucleotide excision repair recognition factor, XPC. Knockdown of NEIL1 in LN428 glioblastoma cells resulted in enhanced recruitment of XPC, a more rapid removal of digoxigenin-tagged psoralen adducts, and decreased cellular sensitivity to trioxsalen plus UVA, implying that NEIL1 and BER may interfere with normal cellular processing of interstrand crosslinks. While exhibiting no enzymatic activity, purified NEIL1 protein bound stably to psoralen interstrand crosslink-containing synthetic oligonucleotide substrates in vitro. Our results indicate that NEIL1 recognizes specifically and distinctly interstrand crosslinks in DNA, and can obstruct the efficient removal of lethal crosslink adducts.
authors.affiliation: Laboratory of Molecular Gerontology, Biomedical Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: McNeill
authors.forename: Manikandan
authors.initials: M
authors.lastname: Paramasivam
authors.forename: Jakita
authors.initials: J
authors.lastname: Baldwin
authors.forename: Jing
authors.initials: J
authors.lastname: Huang
authors.forename: Vaddadi N
authors.initials: VN
authors.lastname: Vyjayanti
authors.forename: Michael M
authors.initials: MM
authors.lastname: Seidman
authors.forename: David M
authors.initials: DM
authors.lastname: Wilson
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000187:drug effects* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005363:Ficusin / Q000494:pharmacology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D016166:Free Radical Scavengers; C086984:XPC protein, human; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; D005363:Ficusin; D000111:Acetylcysteine
keywords: Base Excision Repair; DNA Crosslink; DNA Damage; DNA Damage Response; DNA Repair; NEIL1 Glycosylase; Nonsynonymous Polymorphism; Protein DNA-Interaction; Psoralen