Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster Raghuvar Dronamraju, James M. Mason* Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America Abstract Telomere capture, a rare event that stabilizes chromosome breaks, is associated with certain genetic abnormalities in humans. Studies pertaining to the generation, maintenance, and biological effects of telomere formation are limited in metazoans. A mutation, mu2a, in Drosophila melanogaster decreases the rate of repair of double strand DNA breaks in oocytes, thus leading to chromosomes that have lost a natural telomere and gained a new telomere. Amino acid sequence, domain architecture, and protein interactions suggest that MU2 is an ortholog of human MDC1. The MU2 protein is a component of meiotic recombination foci and localizes to repair foci in S2 cells after irradiation in a manner similar to that of phosphorylated histone variant H2Av. Domain searches indicated that the protein contains an N-terminal FHA domain and a C-terminal tandem BRCT domain. Peptide pull-down studies showed that the BRCT domain interacts with phosphorylated H2Av, while the FHA domain interacts with the complex of MRE11, RAD50, and NBS. A frameshift mutation that eliminates the MU2 BRCT domain decreases the number and size of meiotic phospho-H2Av foci. MU2 is also required for the intra-S checkpoint in eye-antennal imaginal discs. MU2 participates at an early stage in the recognition of DNA damage at a step that is prerequisite for both DNA repair and cell cycle checkpoint control. We propose a model suggesting that neotelomeres may arise when radiation-induced chromosome breaks fail to be repaired, fail to arrest progression through meiosis, and are deposited in the zygote, where cell cycle control is absent and rapid rounds of replication and telomere formation ensue. Citation: Dronamraju R, Mason JM (2009) Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster. PLoS Genet 5(5): e1000473. doi:10.1371/journal.pgen.1000473 Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America Received November 26, 2008; Accepted April 7, 2009; Published May 8, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was funded by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, ES021054. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: masonj@niehs.nih.gov Introduction A single unrepaired DNA double strand break (DSB) in a dividing cell is a potentially lethal event. DSBs are generated naturally upon the collapse of replication fork [1], genome rearrangement by yeast mating type switching [2], V(D)J recombination [3], meiosis [4,5] and exogenous damage. Two main pathways implicated in the repair of a DSB are homologous recombination (HR) and nonhomologous end joining (NHEJ). A cell responds to a DSB by recruiting a host of DNA damage response (DDR) proteins to the chromatin sites near the DSB [6]. While most of the DDR proteins function in either HR or NHEJ, a number of them influence both pathways, including the MRE11/RAD50/NBS1 (MRN) complex, BRCA1, histone H2AX, DNA PKcs and ATM [7–9]. A high degree of conservation in DSB repair systems makes it convenient to use a model organism such as Drosophila to investigate these basic processes. Thus, it may also be possible to obtain and characterize mutations for specific genes necessary for DNA repair. Unrepaired broken chromosome ends are highly unstable, initiating cell cycle arrest or cycling through repeated breakage- fusion-bridge cycles [10]. In the germline they are rarely transmitted to the next generation [11], although occasionally these broken chromosomes are stabilized and are propagated normally [12]. The mechanism of de novo telomere addition to nontelomeric DNA is unclear, but in systems using telomerase to maintain telomeres the enzyme telomerase is known to play an important role [13]. The bulk of our knowledge concerning de novo telomere formation comes from studying ciliates that undergo developmentally programmed chromosome fragmenta- tion and healing [14]. In most organisms, the only evidence for telomere loss is through the detection of aberrant karyotypes including terminal chromosome deficiencies that are successfully propagated as a result of de novo telomere formation. Terminal deficiencies are frequent in tumors [15], as exemplified by malignant melanomas in humans containing deletions of chromo- some 6 [16]. Given the consequences of terminal deficiencies in humans, it would be of interest to study the nature and mechanistic details of their generation and maintenance in a genetically amenable organism such as Drosophila. An ionizing radiation-dependent, female-specific mutator (mu2a) on chromosome 3 of Drosophila melanogaster has been described that specifically increases the recovery of terminal deficiencies, i.e., chromosomes that have lost a natural telomere. Mature mu2a oocytes when treated with 5 Gy of c irradiation show an increase in the frequency of y mutants over the controls. Cytological and molecular analysis of the y mutant chromosomes from mu2a mothers showed that the great majority were terminal deficiencies [17]. mu2a females exhibited a delay in the repair of c ray induced lesions in the oocyte [17], as well as reduced meiotic recombina- tion [18]. Although lesions induced in mature sperm are repaired after fertilization under the genetic control of female to which the PLoS Genetics | www.plosgenetics.org 1 May 2009 | Volume 5 | Issue 5 | e1000473