DNA Repair 10 (2011) 1203–1212 Contents lists available at SciVerse ScienceDirect DNA Repair jo u rn al hom epa ge: www.elsevier.com/locate/dnarepair Human MutS and FANCM complexes function as redundant DNA damage sensors in the Fanconi Anemia pathway Min Huang a , Richard Kennedy b , Abdullah M. Ali c , Lisa A. Moreau a , Amom Ruhikanta Meetei c , Alan D. D’Andrea a , Clark C. Chen a,d,∗ a Department of Radiation Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, MA 02215, United States b Almac Diagnostics, Northern Ireland, UK c Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Research Foundation, University of Cincinnati College of Medicine, Cincinnati, OH 45229, United States d Division of Neurosurgery, Beth Israel Deaconess Medical Center, 110 Francis St., Boston, MA 02115, United States a r t i c l e i n f o Article history: Received 1 June 2011 Received in revised form 3 September 2011 Accepted 10 September 2011 Available online 4 October 2011 Keywords: Fanconi Anemia MutS homologs DNA damage response Sensor a b s t r a c t The Fanconi Anemia (FA) pathway encodes a DNA damage response activated by DNA damage-stalled replication forks. Current evidence suggests that the FA pathway initiates with DNA damage recogni- tion by the FANCM complex (FANCM/FAAP24/MHF). However, genetic inactivation of FANCM in mouse and DT40 cells causes only a partial defect in the FA pathway activation, suggesting the existence of redundant DNA damage sensors. Here we show that the MutS homologs function in this capacity. A RNAi screen revealed that MSH2 silencing caused defective FA pathway activation, as assessed by damage- induced FANCD2 mono-ubiquitination. A similar FA pathway defect was observed with MSH3 or MSH6 silencing. MSH2 depletion caused cellular phenotypes associated with defective FA pathway, includ- ing mitomycin C hypersensitivity and chromosomal instability. Further, silencing of FANCM in MSH2 deficient HEC59 cells caused a more severe FA defect relative to comparable silencing in MSH2 comple- mented HEC59 + Chr2 cells, suggesting redundant functions between MSH2 and FANCM. Consistent with this hypothesis, depletion of MSH2 resulted in defective chromatin localization of the FA core complex upon DNA damage. Further, MSH2 was co-purified and co-immunoprecipitated with FA core complex components. Taken together, our results suggest that human MutS homologs and FANCM complexes function as redundant DNA damage sensors of the FA pathway. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The Fanconi Anemia (FA) DNA damage response pathway con- stitutes a critical component of mammalian DNA repair processes. The pathway was initially defined by virtue of its inactivation in a rare genetic disorder that bears the name, Fanconi Anemia. Sub- sequent characterization of the FA genes uncovered a critical DNA damage response pathway that is activated by DNA damage-stalled replication forks [1]. Activation of the FA pathway involves a com- plex signal transduction cascade with the mono-ubiquitination of the FANCD2/FANCI heterodimer as a key intermediary step [2,3]. Two distinct but interrelated processes are required for this ubiq- uitination. First, a complex composed of eight FA proteins (FANCA, ∗ Corresponding author at: Clinical Neuro-Oncology, Beth Israel Deaconess Medical Center, Department of Radiation Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, MA 02215, United States. Tel.: +1 617 582 8643; fax: +1 617 582 8213. E-mail addresses: clark chen@dfci.harvard.edu, clarkchen@ucsd.edu (C.C. Chen). B, C, E, F, G, L and FAAP100; referred to as the FA core complex) is recruited to the chromatin [4]. The FA core complex serves as an E3 ubiquitin ligase that, in the presence of the E2 protein UBE2T, mono-ubiquitinates the FANCD2/FANCI heterodimer [5–7]. Sec- ond, the ATR kinase is activated by DNA damage [8]. ATR is required for the phosphorylation of several FA proteins, including FANCD2, FANCI, and FANCA. Mutations that disrupted these phosphorylation events impair FANCD2/FANCI mono-ubiquitination [2,9,10]. Given its critical role in the FA pathway activation, the FANCD2/FANCI mono-ubiquitination event is frequently used as a surrogate for FA pathway activation. One of the central questions in FA biology involves the “sensor” protein responsible for detecting the DNA damage. This question was partially answered by the identification of the FANCM gene [11,12]. Among the known FA proteins that function upstream of FANCD2/FANCI, FANCM is the only one that harbors a DNA binding motif [13]. FANCM and its associated proteins, includ- ing FAAP24, MHF1, and MHF2 preferentially bind to branched DNA structures in vitro [14–16]. Upon binding of branched DNA structures, the FANCM complex is proposed to facilitate FANCD2 1568-7864/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2011.09.006