Academic journal article Genetics

Drosophila ATR in Double-Strand Break Repair

Academic journal article Genetics

Drosophila ATR in Double-Strand Break Repair

Article excerpt

ABSTRACT

The ability of a cell to sense and respond to DNA damage is essential for genome stability. An important aspect of the response is arrest of the cell cycle, presumably to allow time for repair. Ataxia telangiectasia mutated (ATM) and ATR are essential for such cell-cycle control, but some observations suggest that they also play a direct role in DNA repair. The Drosophila ortholog of ATR, MEI-41, mediates the DNA damage-dependent G2-M checkpoint. We examined the role of MEI-41 in repair of double-strand breaks (DSBs) induced by P-element excision. We found that mei-41 mutants are defective in completing the later steps of homologous recombination repair, but have no defects in end-joining repair. We hypothesized that these repair defects are the result of loss of checkpoint control. To test this, we genetically reduced mitotic cyclin levels and also examined repair in grp (DmChk1) and lok (DmChk2) mutants. Our results suggest that a significant component of the repair defects is due to loss of MEI-41-dependent cell cycle regulation. However, this does not account for all of the defects we observed. We propose a novel role for MEI-41 in DSB repair, independent of the Chk1/Chk2-mediated checkpoint response.

CELL-CYCLE regulation is an important response to DNA damage. This regulation couples repair with cell-cycle progression to prevent genomic instability following DNA damage. In the DNA damage checkpoint pathway, sensors recognize DNA damage and then stimulate a variety of responses, including phosphorylation of transducers of the checkpoint pathway. These transducers then activate or inactivate effectors that directly inhibit cell-cycle progression, resulting in arrest of the cell cycle to allow time to repair the damage (reviewed in SANCAR et al. 2004).

Ataxia telangiectasia mutated (ATM) and ATR are importantmediators of DNA damage checkpoints. Both regulateDNAdamage-dependent cell-cycle checkpoints at the G1-to-S transition, within S phase, and at the G2-to-M transition (reviewed in SHILOH 2003; SANCAR et al. 2004). In mammals, ATM responds primarily to doublestrand breaks (DSBs) and is therefore activated by ionizing radiation (IR) (CANMAN et al. 1998). In contrast, ATR responds primarily to the presence of ssDNA and is therefore activated by hyperoxia, DNA polymerase inhibitors, and ultraviolet (UV) radiation (CLIBY et al. 1998; WRIGHT et al. 1998; UNSAL-KACMAZ et al. 2002; DAS and DASHNAMOORTHY 2004). Recent findings, however, suggest that the roles of these checkpoints may be more complex than originally proposed. In mammals, there is evidence that ATM and ATR may regulate each other. For example, ATR may be activated by ssDNA that is generated during ATM-dependent repair of DSBs (WANG et al. 2003). Also, ATR is required to maintain the checkpoint initiated by ATM (Brown and Baltimore 2003).

Although there are Drosophila orthologs for both ATM and ATR, theDNAdamage-dependent checkpoint functions reside primarily in the latter, which is encoded by the mei-41 gene. The MEI-41 protein is required to prevent entry into mitosis before completion of replication and for checkpoints induced by DSBs during all phases of the cell cycle (HARI et al. 1995; SIBON et al. 1999; BRODSKY et al. 2000; GARNER et al. 2001; JAKLEVIC and SU 2004; BI et al. 2005a). As a consequence, mei-41 mutants are hypersensitive to agents that inhibit or block replication, such as hydroxyurea, alkylating chemicals, and ultraviolet radiation, and to agents that generate DSBs, such as IR (BOYD et al. 1976; SIBON et al. 1999). In contrast, the primary roles of the Drosophila ortholog of ATM (encoded by tefu) are in telomere stabilization and regulation of p53-dependent apoptosis; null mutations in tefu are lethal, possibly due to chromosome breakage resulting from telomere fusions (BI et al. 2004, 2005b; SILVA et al. 2004; SONG et al. 2004). Temperature-sensitive tefumutants are hypersensitive to IR, but they have a fully functional checkpoint at high doses of IR (SILVA et al. …

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