Summary Chromosome ends, known as telomeres, have to be distinguished from DNA double-strand breaks (DSBs) that activate the DNA damage checkpoint. In budding yeast, the ATM homolog Tel1 associates preferentially with short telomeres and promotes telomere addition. Here we show that the telomeric proteins Rif1 and Rif2 attenuate Tel1 recruitment to DNA ends through distinct mechanisms. Both Rif1 and Rif2 inhibit the localization of Tel1, but not the Mre11-Rad50-Xrs2 (MRX) complex, to adjacent DNA ends. Rif1 function is weaker at short telomeric repeats compared with Rif2 function, and is partly dependent on Rif2. Rif2 competes with Tel1 for binding to the C-terminus of Xrs2. Once Tel1 is delocalized, MRX does not associate efficiently with Rap1-covered DNA ends. These results reveal a mechanism by which telomeric DNA sequences mask DNA ends from Tel1 recognition for the regulation of telomere length.

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Canonical aversive long-term memory (LTM) formation in Drosophila requires multiple spaced trainings, whereas appetitive LTM can be formed after a single training. Appetitive LTM requires fasting prior to training, which increases motivation for food intake. However, we found that fasting facilitated LTM formation in general; aversive LTM formation also occurred after single-cycle training when mild fasting was applied before training. Both fasting-dependent LTM (fLTM) and spaced training-dependent LTM (spLTM) required protein synthesis and cyclic adenosine monophosphate response element-binding protein (CREB) activity. However, spLTM required CREB activity in two neural populations--mushroom body and DAL neurons--whereas fLTM required CREB activity only in mushroom body neurons. fLTM uses the CREB coactivator CRTC, whereas spLTM uses the coactivator CBP. Thus, flies use distinct LTM machinery depending on their hunger state.

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). Here we characterize global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Bx. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and we identify different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, we find that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. Our results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs.

DNA polymerase zeta (Polzeta) and Rev1 contribute to the bypassing of DNA lesions, termed translesion DNA synthesis (TLS). Polzeta consists of two subunits, one encoded by REV3 (the catalytic subunit) and the other encoded by REV7. Rev1 acts as a deoxycytidyl transferase, inserting dCMP opposite lesions. Polzeta and Rev1 have been shown to operate in the same TLS pathway in the budding yeast Saccharomyces cerevisiae. Here, we show that budding yeast Polzeta and Rev1 form a complex and associate together with double-strand breaks (DSBs). As a component of the Polzeta-Rev1 complex, Rev1 plays a noncatalytic role in the association with DSBs. In budding yeast, the ATR-homolog Mec1 plays a central role in the DNA-damage checkpoint response. We further show that Mec1-dependent phosphorylation promotes the Polzeta-Rev1 association with DSBs. Rev1 association with DSBs requires neither the function of the Rad24 checkpoint-clamp loader nor the Rad6-Rad18-mediated ubiquitination of PCNA. Our results reveal a novel role of Mec1 in the localization of the Polzeta-Rev1 complex to DNA lesions and highlight a linkage of TLS polymerases to the checkpoint response.

Key points• During olfactory aversive conditioning in Drosophila, odour and shock information are delivered to the mushroom bodies (MBs) through projection neurons in the antennal lobes (ALs) and ascending fibres of the ventral nerve cord (AFV), respectively.• Using an isolated cultured brain expressing a Ca 2+ indicator in the MBs, we demonstrated that the simultaneous stimulation of the ALs and AFV establishes long-term enhancement (LTE) in AL-induced Ca 2+ responses.• The physiological properties of LTE, including associativity, input specificity and persistence, are highly reminiscent of those of olfactory memory.• Similar to olfactory aversive memory, LTE requires the activation of nicotinic acetylcholine receptors that mediate the AL-evoked Ca 2+ response, NMDA receptors that mediate the AFV-induced Ca 2+ response, and D1 dopamine receptors during the simultaneous stimulation of the ALs and AFV.• Considering the physiological and genetic analogies, we propose that LTE at the AL-MB synapse can be a relevant cellular model for olfactory memory.Abstract In Drosophila, the mushroom body (MB) is a critical brain structure for olfactory associative learning. During aversive conditioning, the MBs are thought to associate odour signals, conveyed by projection neurons (PNs) from the antennal lobe (AL), with shock signals conveyed through ascending fibres of the ventral nerve cord (AFV). Although synaptic transmission between AL and MB might play a crucial role for olfactory associative learning, its physiological properties have not been examined directly. Using a cultured Drosophila brain expressing a Ca 2+ indicator in the MBs, we investigated synaptic transmission and plasticity at the AL-MB synapse.Following stimulation with a glass micro-electrode, AL-induced Ca 2+ responses in the MBs were mediated through Drosophila nicotinic acetylcholine receptors (dnAChRs), while AFV-induced Ca 2+ responses were mediated through Drosophila NMDA receptors (dNRs). AL-MB synaptic transmission was enhanced more than 2 h after the simultaneous 'associative-stimulation' of AL and AFV, and such long-term enhancement (LTE) was specifically formed at the AL-MB synapses but not at the AFV-MB synapses. AL-MB LTE was not induced by intense stimulation of the AL alone, and the LTE decays within 60 min after subsequent repetitive AL stimulation. These phenotypes of associativity, input specificity and persistence of AL-MB LTE are highly reminiscent of olfactory memory. Furthermore, similar to olfactory aversive memory, AL-MB LTE formation required activation of the Drosophila D1 dopamine receptor, DopR, along with dnAChR and dNR

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), coordinate the cellular response to DNA damage. In budding yeast, ATR homologue Mec1 plays a central role in DNA damage signaling. Mec1 interacts physically with Ddc2 and functions in the form of the Mec1-Ddc2 complex. To identify proteins interacting with the Mec1-Ddc2 complex, we performed a modified two-hybrid screen and isolated RFA1 and RFA2, genes that encode subunits of replication protein A (RPA). Using the two-hybrid system, we found that the extreme C-terminal region of Mec1 is critical for RPA binding. The C-terminal substitution mutation does not affect the Mec1-Ddc2 complex formation, but it does impair the interaction of Mec1 and Ddc2 with RPA as well as their association with DNA lesions. The C-terminal mutation also decreases Mec1 kinase activity. However, the Mec1 kinase-defect by itself does not perturb Mec1 association with sites of DNA damage. We also found that Mec1 and Ddc2 associate with sites of DNA damage in an interdependent manner. Our findings support the model in which Mec1 and Ddc2 localize to sites of DNA damage by interacting with RPA in the form of the Mec1-Ddc2 complex. INTRODUCTIONThe maintenance of genome stability is critical to cellular survival and proliferation in all organisms. Cells have evolved surveillance mechanisms that monitor genomic lesions and activate various DNA damage responses, including cell cycle arrest and transcriptional induction of DNA repair genes (Zhou and Elledge, 2000). This surveillance mechanism is called DNA damage checkpoint in eukaryotes. The checkpoint signals are initiated through two large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) (Zhou and Elledge, 2000;Abraham, 2001). ATM and ATR are highly conserved among eukaryotes. ATR is closely related to Mec1 in the budding yeast Saccharomyces cerevisiae and Rad3 in the fission yeast Schizosaccharomyces pombe. ATM homologues are termed Tel1 in both budding and fission yeasts.In the budding yeast S. cerevisiae, Mec1 plays a central role in DNA damage checkpoint control, whereas Tel1 plays a minor role (Morrow et al., 1995;Sanchez et al., 1996;Usui et al., 2001;Nakada et al., 2003bNakada et al., , 2004. Mec1 physically interacts with Ddc2 (also called Lcd1 and Pie1), a protein that exhibits homology to the ATR-interacting protein ATRIP and Rad3-interacting protein Rad26 (Edwards et al., 1999;Paciotti et al., 2000;Rouse and Jackson, 2000;Cortez et al., 2001;Wakayama et al., 2001). Mec1 and Ddc2 function in the form of the Mec1-Ddc2 complex, and both localize to sites of DNA damage Melo et al., 2001;Rouse and Jackson, 2002). Mec1 controls two downstream protein kinases Chk1 and Rad53, which are related to mammalian Chk1 and Chk2, respectively (Zhou and Elledge, 2000). Rad53 plays a central role in DNA damage checkpoints throughout the cell cycle (Longhese et al., 1998), whereas Chk1 acts in part at G 2 /M (Sanchez et al., 1999). Rad53 becomes phosphorylated and activated after ...

Several aging phenotypes, including age-related memory impairment (AMI), are thought to be caused by cumulative oxidative damage. In Drosophila, age-related impairments in 1 hr memory can be suppressed by reducing activity of protein kinase A (PKA). However, the mechanism for this effect has been unclear. Here we show that decreasing PKA suppresses AMI by reducing activity of pyruvate carboxylase (PC), a glial metabolic enzyme whose amounts increase upon aging. Increased PC activity causes AMI through a mechanism independent of oxidative damage. Instead, increased PC activity is associated with decreases in D-serine, a glia-derived neuromodulator that regulates NMDA receptor activity. D-serine feeding suppresses both AMI and memory impairment caused by glial overexpression of dPC, indicating that an oxidative stress-independent dysregulation of glial modulation of neuronal activity contributes to AMI in Drosophila.