Volume 35, Issue 5, Pages 716-723 (September 2009) Recruitment of Fanconi Anemia and Breast Cancer Proteins to DNA Damage Sites Is Differentially Governed by Replication  Xi Shen, Huong Do, Yongjiang Li, Woo-Hyun Chung, Maria Tomasz, Johan P. de Winter, Bing Xia, Stephen J. Elledge, Weidong Wang, Lei Li  Molecular Cell  Volume 35, Issue 5, Pages 716-723 (September 2009) DOI: 10.1016/j.molcel.2009.06.034 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 eChIP Assay for the In Vivo Identification of Proteins Recruited to a Defined Interstrand Crosslink (A) Schematics of the pORIP DNA substrate under replicative and nonreplicative states. Short bars adjacent to the DNA interstrand crosslink indicate the region of PCR amplification. (B) Replication competence of the pORIP substrate. Unmodified pORIP vector was transiently introduced into and allowed to replicate in HEK293 and HEK293-EBNA cells, respectively. Dpn1-resistant colonies were measured for each cell line. Error bars reflect STDEV from three independent experiments. (C) Endogenous FANCD2 ubiquitination induced by crosslinked, ectopically introduced pORIP substrate. HEK239 and HEK239-EBNA cells were transfected with crosslinked (XL) and unmodified (Ctrl) pORIP substrates and harvested at 3 and 6 hr. Immunoblotting was performed with an anti-FANCD2 antibody. β-tubulin was used as a loading control. (D) Replication-dependent enrichment of MCM7 at a site-specific psoralen crosslink as detected by ChIP. Inverted agarose gel image is shown in this and ensuing experiments employing the eChIP assay. (E) Quantified relative enrichment of MCM7 at a crosslink as derived from (D). Molecular Cell 2009 35, 716-723DOI: (10.1016/j.molcel.2009.06.034) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Differential Recruitment of Fanconi Anemia Proteins to a Defined Interstrand Crosslink as a Function of DNA Replication eChIP analyses were carried out in the presence (293-EBNA) or absence (293) of DNA replication. (A) Crosslinking-dependent recruitment of FAAP24, FANCJ, and FANCA. (B) Crosslinking-dependent recruitment of FANCI and FANCN. (C) Crosslinking-dependent recruitment of FANCD2 and FANCC. (D) Crosslinking-dependent recruitment of FANCD1. (E) IP-western examining the interaction of EBNA-1 with FANCD2, FANCJ, and RPA70 in four types of cell lysates: 293, 293 transfected with pORIP, 293EBNA, and 293EBNA transfected with pORIP. Molecular Cell 2009 35, 716-723DOI: (10.1016/j.molcel.2009.06.034) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Recruitment of Fanconi Anemia Proteins in Wild-Type and FANCA Mutant Cells A wild-type lymphoblastoid cell line (WT) ManEBV was used as a positive control. (A) Immunoblotting of the FANCA mutant VU388 (FANCA−) and its complimented derivative (FANCA+) with an anti-FANCA antibody. β-tubulin was used to control for loading. (B) Recruitment of FAAP24 and FANCD2 in WT, FANCA+, and FANCA− cells. (C) Recruitment of FANCN in FANCA+ and FANCA− cells. (D) Recruitment of FANCJ, WT, FANCA+, and FANCA− cells. Molecular Cell 2009 35, 716-723DOI: (10.1016/j.molcel.2009.06.034) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Components of the Canonical FA Proteins Are Linked to Recombination-Independent Crosslink Repair (A) Replication-independent recruitment of FANCA to defined interstrand crosslinks in XPA and XPC mutant cells. (B) Recombination-independent repair of crosslinks in FANCD2 and FANCJ cells measured by the luciferase reactivation assay. ICL repair efficiency of each cell line was arrived at by normalizing the luciferase activity from parallel seeded cells transfected with crosslinked luciferase plasmid against that of cells transfected with an unmodified luciferase plasmid. The luciferase activity of each sample was also normalized against an internal β-gal control. Error bars were derived from multiple experiments with duplicated transfections. (C) Models depicting differential recruitment of FANC proteins and its potential implications. (Left) Recruitment of FANC core and ID complexes by crosslinks in the absence of DNA replication. (Right) Replication-dependent recruitment of BRCA-related FANC proteins. Molecular Cell 2009 35, 716-723DOI: (10.1016/j.molcel.2009.06.034) Copyright © 2009 Elsevier Inc. Terms and Conditions