title: 3CAPS - a structural AP-site analogue as a tool to investigate DNA base excision repair.
abstract: Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3'-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5'-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.
authors.affiliation: Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland david.schuermann@unibas.ch.
authors.forename: David
authors.initials: D
authors.lastname: Schuermann
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: Simon P
authors.initials: SP
authors.lastname: Scheidegger
authors.affiliation: Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland.
authors.forename: Alain R
authors.initials: AR
authors.lastname: Weber
authors.affiliation: Department of Microbiology, Oslo University Hospital and University of Oslo, Rikshospitalet, PO Box 4950 Nydalen, N-0424 Oslo, Norway Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, PO Box 8905, N-7491 Trondheim, Norway.
authors.forename: Magnar
authors.initials: M
authors.lastname: Bjørås
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: Christian J
authors.identifier: http://orcid.org/0000-0002-7996-7083
authors.initials: CJ
authors.lastname: Leumann
authors.affiliation: Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland primo.schaer@unibas.ch.
authors.forename: Primo
authors.initials: P
authors.lastname: Schär
mesh_terms: D000080:Acetals / Q000737:chemistry / Q000378:metabolism; D001681:Biological Assay; D040761:Biomimetic Materials / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000080:Acetals; D009841:Oligonucleotides; D011994:Recombinant Proteins; D004247:DNA; D019951:DNA Polymerase beta; D043224:Deoxyribonuclease (Pyrimidine Dimer); C468347:NTHL1 protein, human; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase