Abstract
Chromatin structure regulates the dynamics of the recognition and repair of DNA double strand breaks; open chromatin enhances the recruitment of DNA damage response factors, while compact chromatin is refractory to the assembly of radiation-induced repair foci. MU2, an orthologue of human MDC1, a scaffold for ionizing radiation-induced repair foci, is a widely distributed chromosomal protein in Drosophila melanogaster that moves to DNA repair foci after irradiation. Here we show using yeast 2 hybrid screens and co-immunoprecipitation that MU2 binds the chromoshadow domain of the heterochromatin protein HP1 in untreated cells. We asked what role HP1 plays in the formation of repair foci and cell cycle control in response to DNA damage. After irradiation repair foci form in heterochromatin but are shunted to the edge of heterochromatic regions an HP1-dependent manner, suggesting compartmentalized repair. Hydroxyurea-induced repair foci that form at collapsed replication forks, however, remain in the heterochromatic compartment. HP1a depletion in irradiated imaginal disc cells increases apoptosis and disrupts G2/M arrest. Further, cells irradiated in mitosis produced more and brighter repair foci than to cells irradiated during interphase. Thus, the interplay between MU2 and HP1a is dynamic and may be different in euchromatin and heterochromatin during DNA break recognition and repair.
Highlights
Homologous recombination and nonhomologous end joining are the two major mechanisms for repair of double strand breaks (DSBs) in DNA, ensuring the transmission of intact genetic information
Four fragments that included the region of MU2 preceding the tandem BRCA1 C-terminal (BRCT) domain showed a large number of hits, but no hits were found with other fragments, suggesting a possible interaction between MU2 and Heterochromatin protein 1a (HP1a) in this domain
These results indicate that this region of MU2 interacts with the chromoshadow domain of HP1a, which is a well-established domain for protein-protein interactions
Summary
Homologous recombination and nonhomologous end joining are the two major mechanisms for repair of double strand breaks (DSBs) in DNA, ensuring the transmission of intact genetic information. DSBs induce cellular signals, which are primarily dependent upon the activation of the ATM kinase and culminate in the recruitment of DNA damage response (DDR) proteins to the break. Phosphorylation of H2AX (Drosophila homolog: H2Av) to produce cH2AX is one of the earliest chromatin modifications that sets the stage for the assembly of multi-protein complexes that are microscopically discernible as foci [4,5,6]. Extensive genetic analysis suggested that MU2 may be a chromatin protein and play an important role in the repair of radiation-induced DSBs [10]. The polytene chromosomes are covered with MU2 in a pattern similar to DAPI staining. A striking feature of the protein is the presence of a C-
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