DNA Repair 8 (2009) 1225–1234 Contents lists available at ScienceDirect DNA Repair journal homepage: www.elsevier.com/locate/dnarepair DNA lesions sequestered in micronuclei induce a local defective-damage response Mariona Terradas, Marta Martín, Laura Tusell, Anna Genescà ∗ Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain article info Article history: Received 3 April 2009 Received in revised form 10 July 2009 Accepted 13 July 2009 Available online 14 August 2009 Keywords: DNA damage response Micronuclei H2AX Apoptosis Senescence abstract Micronuclei are good markers of chromosome instability and, among other disturbances, are closely related to double-strand break induction. The ability of DNA lesions sequestered in the micronuclear bod- ies to activate the complex damage-signalling network is highly controversial since some repair factors have not been consistently detected inside micronuclei. In order to better understand the efficiency of the response induced by micronuclear DNA damage, we have analyzed the presence of DNA damage-response factors and DNA degradation markers in these structures. Radiation-induced DNA double-strand breaks produce a modification of chromatin structural proteins, such as the H2AX histone, which is rapidly phos- phorylated around the break site. Strikingly, we have been able to distinguish two different phosphoH2AX (H2AX) labelling patterns in micronuclei: discrete foci, indicating DSB presence, and uniform labelling affecting the whole micronucleus, pointing to genomic DNA fragmentation. At early post-irradiation times we observed a high fraction of micronuclei displaying H2AX foci. Co-localization experiments showed that only a small fraction of the DSBs in micronuclei were able to properly recruit the p53 binding pro- tein 1 (53BP1) and the meiotic recombination 11 (MRE11). We suggest that trafficking defects through the micronuclear envelope compromise the recruitment of DNA damage-response factors. In contrast to micronuclei displaying H2AX foci, we observed that micronuclei showing a H2AX extensive-uniform labelling were more frequently observed at substantial post-irradiation times. By means of TUNEL assay, we proved that DNA degradation was carried out inside these micronuclei. Given this scenario, we pro- pose that micronuclei carrying a non-repaired DSB are conduced to their elimination, thus favouring chromosome instability in terms of allele loss. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Damaged DNA efficiently activates a complex signalling network known as DNA damage response (DDR), which coordinates tem- porary delay in cell-cycle progression and repair machinery. After irradiation, the cytological manifestation of the allocation of DDR components to regions containing damaged DNA is the formation of, what is termed, IR-induced foci (IRIF) [1]. IRIF are dynamic and microscopically visible discrete structures containing thousands of copies of the proteins involved in the DNA double-strand break (DSB) metabolism. After a DSB is detected, one of the first steps to occur in the damaged cell is the phosphorylation of H2AX on its Ser139 [2], which can affect as much as one megabase of the chromatin flanking the DSB [3]. Phosphorylated H2AX spreading is considered the platform through which DSB recognition fac- tors, chromatin-modifying proteins and checkpoint proteins are recruited and retained to allow DSB repair [4–8]. When damage in the cell nucleus is irreparable, the DDR activates apoptotic DNA degradation or cell senescence. Histone H2AX is also phosphory- ∗ Corresponding author. Tel.: +34 935811498; fax: +34 935812295. E-mail address: anna.genesca@uab.cat (A. Genescà). lated on its Ser139 during the DNA degradation that characterizes apoptosis [9]. H2AX is therefore commonly used as a marker for the presence of DNA DSBs and apoptotic genome fragmentation in the cell nucleus. The importance of effective DDR in order to maintain the cell genomic stability is extensively recognized. When these mech- anisms fail, cells become genomically instable and chromosome aberrations and mutations are generated continuously. One of the main forms of genomic instability is at the chromosome level, and chromosomal instability, a transient or persistent state character- ized by an accelerated rate of structural and numerical chromosome aberration formation, is a key component in tumorigenesis [10,11]. The connection between chromosomal instability and tumorigen- esis relies on gene loss and amplification that alter dosages of key oncogenic proteins. Chromosomal instability results from repeated series of anaphase bridge breakage and mis-repair of fragments, resulting in the formation of new bridges, a mechanism known as breakage–fusion-bridge (BFB) cycles. These cycles can be initiated by DNA breaks [12], abnormal shortening of telomeres [13] or per- sistent chromatid cohesion [14,15]. Broken bridges can result in the formation of micronuclei and nuclear blebs at the end of mitosis [16,17]. Abnormal nuclear morphologies are considered good indi- cators of chromosome instability, as they have been observed in a 1568-7864/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2009.07.004