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Molecular and Cellular Biology, December 2009, p. 6427-6437, Vol. 29, No. 24
0270-7306/09/$08.00+0     doi:10.1128/MCB.00086-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

XPF-ERCC1 Participates in the Fanconi Anemia Pathway of Cross-Link Repair {triangledown}

Nikhil Bhagwat,1,2,{dagger} Anna L. Olsen,3,{dagger} Anderson T. Wang,3 Katsuhiro Hanada,4 Patricia Stuckert,5 Roland Kanaar,4,6 Alan D'Andrea,5 Laura J. Niedernhofer,2,7* and Peter J. McHugh3*

Department of Human Genetics, University of Pittsburgh School of Public Health, A300 Crabtree Hall, 130 Desoto St., Pittsburgh, Pennsylvania 15261,1 University of Pittsburgh Cancer Institute, Hillman Cancer Center, Research Pavilion 2.6, 5117 Centre Ave., Pittsburgh, Pennsylvania 15213-1863,2 Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom,3 Department of Cell Biology & Genetics, Cancer Genomics Center, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands,4 Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney St., Boston, Massachusetts 02115,5 Department of Radiation Oncology, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands,6 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, E1240 BSTWR, 200 Lothrop St., Pittsburgh, Pennsylvania 152617

Received 19 January 2009/ Returned for modification 28 February 2009/ Accepted 26 September 2009

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81–/–, Ercc1–/–, and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

* Corresponding author. Mailing address for Peter J. McHugh: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Phone: 44-1865-222441. Fax: 44-1865-222431. E-mail: peter.mchugh{at}imm.ox.ac.uk. Mailing address for Laura J. Niedernhofer: University of Pittsburgh Cancer Institute, Hillman Cancer Center, Research Pavilion 2.6, 5117 Centre Ave., Pittsburgh, PA 15213-1863. Phone: (412) 623-7763. Fax: (412) 623-7761. E-mail: niedernhoferl{at}upmc.edu

{triangledown} Published ahead of print on 5 October 2009.

{dagger} These authors contributed equally.

Molecular and Cellular Biology, December 2009, p. 6427-6437, Vol. 29, No. 24
0270-7306/09/$08.00+0     doi:10.1128/MCB.00086-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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