DNA Repair 3 (2004) 535–542 Interstrand crosslink-induced radials form between non-homologous chromosomes, but are absent in sex chromosomes Amy E. Hanlon Newell a , Yassmine M.N. Akkari a , Yumi Torimaru a , Andrew Rosenthal a , Carol A. Reifsteck a , Barbara Cox a , Markus Grompe a,b , Susan B. Olson a,∗ a Department of Molecular and Medical Genetics, Oregon Health & Science University, MP350, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA b Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239, USA Received 10 October 2003; accepted 27 January 2004 Abstract Fanconi anemia (FA) and cells lacking functional BRCA1 and BRCA2 proteins are hypersensitive to interstrand crosslinking (ICL) agents and show increased numbers of chromosomal breaks and radials. Although radial formation has been used to diagnose FA for more than 30 years, there has been little analysis of these characteristic formations. In this study, radials were analyzed from FA-A and FA-G fibroblasts as well as normal and retrovirally-corrected FA-A fibroblasts treated with high doses of ICLs. Radials were found to only involve non-homologous chromosome interactions and to be distributed nearly randomly along the length of chromosomes. Sites on chromosomes that did show increased frequency of radial involvement did not correlate with known fragile sites or pericentric regions. Hybrid radials were observed between mouse and human chromosomes in human–mouse hybrid cells produced by microcell-mediated chromosome transfer of mouse chromosomes into human FA-A fibroblasts. Both X and Y chromosomes were notably not involved in radials. These observations suggest that ICL repair may involve short stretches of homology, resulting in aberrant radial formation in the absence of FA proteins. © 2004 Elsevier B.V. All rights reserved. Keywords: Chromosomes; Fanconi anemia; Interstrand crosslink 1. Introduction DNA interstrand crosslinks (ICLs) form as covalent bonds between two nucleotides on the two opposite DNA strands when cells are treated with chemicals such as mitomycin C (MMC),diepoxybutane (DEB), and photoactivated pso- ralens [1]. This covalent bond presents a challenge for the cellular repair machinery since there is no complementary strand available as a template. Recognition of ICLs has been shown to occur during S phase [2]; therefore, repair of these lesions is likely to be attempted following replication. This is consistent with the formation of sister chromatid breaks and radials following ICL damage which are visible cyto- genetically at metaphase. ∗ Corresponding author. Tel.:+1-503-494-5964; fax: +1-503-494-6104. E-mailaddress: olsonsu@ohsu.edu (S.B. Olson). Much of the research addressing ICL repair in mammalian cells has focused on Fanconi anemia (FA) cells which are hypersensitive to ICL agents [3,4]. FA cells form radials at much lower concentrations of MMC than wild-type cells [5] and display a significant cellcycle delay with a 4N DNA content after introduction of ICLs [6]. Taking advantage of this hypersensitivity, clinical diagnosis is conducted by ex- posing cells from FA patients to ICL agents such as MMC and DEB and scoring for the formation of breaks and radials [7,8] (Fig. 1). Additionally, this unique cellular phenotype is used in complementation studies and other experiments that explore the FA genes and their function [9–11]. Although the understanding of FA proteinsand their function is continually increasing, the molecular mecha- nism of ICL repair in mammals is yet to be elucidated. It has been proposed that mammalian ICL repair may involve non-homologous end-joining, homologous recombination, or both [12].Recently, BRCA1 and BRCA2, known to be involved in homologous recombination and maintenance of 1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.01.011