no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila

Julie A. Merkle Jamie L. Rickmyre Aprajita Garg Erin B. Loggins Jeanne N. Jodoin Ethan Lee Louisa P. Wu Laura A. Lee In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We identified CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, as the nopo gene. A conserved residue in the RING domain is altered in our EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. We show that mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. We show that Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitination complex consisting of BEN-UEV1A (E2 heterodimer) and NOPO (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis. INTRODUCTION To ensure faithful transmission of the genome upon cell division, eukaryotic cells have developed checkpoints, regulatory pathways that delay cell-cycle progression until completion of prior events. The DNA damage/replication checkpoint plays a crucial role in preserving genomic integrity (Branzei and Foiani, 2008). Upon detection of DNA defects, the kinases ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3-related) are recruited to sites of damage and activated. ATM and ATR substrates include checkpoint kinases CHK1 and CHK2, which phosphorylate proteins that mediate cell-cycle arrest. The ensuing delay, resulting from engagement of this checkpoint, presumably allows cells time to correct defects. Research over the past decade has highlighted major roles for protein ubiquitination in regulating cellular responses to DNA damage (Harper and Elledge, 2007). This post-translational modification, which involves covalent linkage of one or more ubiquitin molecules to another protein, regulates many fundamental cellular processes (Pickart, 2001). Ubiquitination may alter the fate of a protein in numerous ways, such as targeting it for destruction by the 26S proteasome, changing its subcellular location, or changing its protein-protein interactions. Ubiquitination is a highly dynamic, multi-step process that requires three components: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2 or Ubc) and ubiquitin ligase (E3). E3s can be divided into two main classes: HECT and RING domaincontaining proteins. RING-type E3 ubiquitin ligases (Freemont, 2000; Jackson et al., 2000) contain a specialized motif of 40 to 60 residues that binds two zinc atoms. Many RING-type E3s bind to partnering E2 conjugating enzymes via their RING domains (Passmore and Barford, 2004). Database searches of the Drosophila genome predict that it contains one E1, 36 E2s and ~130 E3s, which represents ~40% of the ubiquitination machinery in humans (Ditzel and Meier, 2005). Significant insights into the roles of many cell-cycle regulators have come from studying their functions in Drosophila. Drosophila is well suited for studying cell-cycle regulation during the formation of a multicellular organism, in large part because of its developmental use of cell cycles that differ in structure from canonical G1SG2M cycles and the availability of genetic tools (Garcia et al., 2007; Lee and Orr-Weaver, 2003). The first thirteen cell cycles of Drosophila embryogenesis involve nearly synchronous nuclear divisions driven by stockpiles of maternally expressed mRNA and protein (Foe et al., 1993). These rapid cycles (~10 minutes in length) consist of oscillating SM (DNA replicationmitosis) phases without intervening gap phases or cytokinesis. Minimal gene transcription occurs during this developmental stage, so cell cycles are regulated by post-transcriptional mechanisms. At cycle 14, the embryo cellularizes and initiates zygotic transcription at the midblastula transition (MBT). We report here the identification and characterization of a Drosophila maternal-effect lethal mutant that we have named no poles (nopo). Embryos from nopo females undergo mitotic arrest with acentrosomal, barrel-shaped spindles during syncytial divisions. Our results indicate that this arrest is secondary to the activation of a CHK2-mediated DNA checkpoint in early embryos. We show that NOPO, a predicted E3 ubiquitin ligase, interacts with an E2 component, BEN. ben females are sterile, producing embryos with nopo-like defects. We propose that BEN-UEV1A and NOPO function together as an E2-E3 complex required for genomic integrity during Drosophila embryogenesis. MATERIALS AND METHODS Drosophila stocks Flies were maintained at 25C using standard techniques. y w was used as wild type unless otherwise indicated. cn Z2-1447 bw/CyO was a gift from Charles Zuker (UC San Diego); ben1 and mnk6006 stocks were from Mark Tanouye (UC Berkeley) and Bill Theurkauf (UMass Worcester), respectively; and the EYG5845 stock was from GenExel (Seoul, Korea). Other fly stocks were from the Bloomington or Szeged stock centers. Quantification of egg hatch rates Five newly eclosed females of the indicated genotype and five wild-type males were incubated in yeast-pasted vials for two days and transferred to egg-collection chambers at 25C. Eggs were collected daily over five days and scored for hatching ~40 hours post-collection (>500 eggs per genotype). Hatch rate is the ratio of hatched to total eggs expressed as a percentage. Genetic and molecular mapping of nopo We screened a second chromosome deficiency collection for noncomplementation of female sterility of nopoZ1447. Females carrying nopoZ1447 in trans to any of several overlapping deficiencies (Df(2R)Pcl-11B, Df(2R)Pcl-XM82, Df(2R)Pcl-7B or Df(2R)PC4) were sterile, placing nopo in the 55A1-C1 interval. We further mapped nopoZ1447 by P-element-induced male recombination (Chen et al., 1998) relative to several insertions: lolalEP2169, Dgp-1BG00396, CG5721EY03388, fjKG03419 and EP(2)1081. Multiple independent recombinant chromosomes were recovered for each P-element tested. We narrowed nopo to five candidates in the 55B11-12 region (Dgp-1, CG10916, CG5726, CG5140 and CG5721) distal to Dgp-1BG00396 and proximal to CG5721EY03388, as annotated on FlyBase (Grumbling and Strelets, (...truncated)