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FANCD2-associated nuclease 1, but not exonuclease 1 or flap endonuclease 1, is able to unhook DNA interstrand cross-links in vitro


Pizzolato, Julia; Mukherjee, Shivam; Schaerer, Orlando D; Jiricny, Josef (2015). FANCD2-associated nuclease 1, but not exonuclease 1 or flap endonuclease 1, is able to unhook DNA interstrand cross-links in vitro. Journal of Biological Chemistry, 290:22602-22611.

Abstract

Cisplatin and its derivatives, nitrogen mustards (NMs) and mitomycin C (MMC) are widely used 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 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 3-4 nucleotides in the double-stranded region. This affected the outcome of processing of a substrate containing an NM-like ICL two nucleotides in the duplex region, because FAN1, unlike EXO1 and FEN1, incised the substrate predominantly beyond the ICL and thus 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 elucidate its biological function, which is currently unknown.

Abstract

Cisplatin and its derivatives, nitrogen mustards (NMs) and mitomycin C (MMC) are widely used 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 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 3-4 nucleotides in the double-stranded region. This affected the outcome of processing of a substrate containing an NM-like ICL two nucleotides in the duplex region, because FAN1, unlike EXO1 and FEN1, incised the substrate predominantly beyond the ICL and thus 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 elucidate its biological function, which is currently unknown.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:28 July 2015
Deposited On:02 Sep 2015 15:58
Last Modified:26 Aug 2016 07:56
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
Additional Information:This research was originally published in The Journal of Biological Chemistry. Pizzolato J et al: FANCD2-associated Nuclease 1, but Not Exonuclease 1 or Flap Endonuclease 1, Is Able to Unhook DNA Interstrand Cross-links in Vitro, The Journal of Biological Chemistry, 2015, 290:22602-22611 © the American Society for Biochemistry and Molecular Biology.
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/jbc.M115.663666
PubMed ID:26221031

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