title: FANCD2-associated nuclease 1, but not exonuclease 1 or flap endonuclease 1, is able to unhook DNA interstrand cross-links in vitro.
abstract: Cisplatin and its derivatives, nitrogen mustards and mitomycin C, are used widely in cancer chemotherapy. Their efficacy is linked primarily to their ability to generate DNA interstrand cross-links (ICLs), which effectively block the progression of transcription and replication machineries. Release of this block, referred to as unhooking, has been postulated to require endonucleases that incise one strand of the duplex on either side of the ICL. Here we investigated how the 5' flap nucleases FANCD2-associated nuclease 1 (FAN1), exonuclease 1 (EXO1), and flap endonuclease 1 (FEN1) process a substrate reminiscent of a replication fork arrested at an ICL. We now show that EXO1 and FEN1 cleaved the substrate at the boundary between the single-stranded 5' flap and the duplex, whereas FAN1 incised it three to four nucleotides in the double-stranded region. This affected the outcome of processing of a substrate containing a nitrogen mustard-like ICL two nucleotides in the duplex region because FAN1, unlike EXO1 and FEN1, incised the substrate predominantly beyond the ICL and, therefore, failed to release the 5' flap. We also show that FAN1 was able to degrade a linear ICL substrate. This ability of FAN1 to traverse ICLs in DNA could help to elucidate its biological function, which is currently unknown.
authors.affiliation: From the Institute of Molecular Cancer Research, University of Zurich and.
authors.forename: Julia
authors.initials: J
authors.lastname: Pizzolato
authors.affiliation: the Departments of Chemistry and.
authors.forename: Shivam
authors.initials: S
authors.lastname: Mukherjee
authors.affiliation: the Departments of Chemistry and Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794-3400.
authors.forename: Orlando D
authors.initials: OD
authors.lastname: Schärer
authors.affiliation: From the Institute of Molecular Cancer Research, University of Zurich and the Department of Biology, Swiss Institute of Technology (ETH) Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland, and jiricny@imcr.uzh.ch.
authors.forename: Josef
authors.initials: J
authors.lastname: Jiricny
mesh_terms: D002460:Cell Line; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D064251:Multifunctional Enzymes; D013379:Substrate Specificity
chemical_list: D064251:Multifunctional Enzymes; D004247:DNA; C114331:EXO1 protein, human; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C551063:FAN1 protein, human; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D045643:DNA Repair Enzymes
keywords: DNA damage; DNA endonuclease; DNA repair; DNA-protein interaction; enzyme mechanism; exonuclease; interstrand cross-links