Mutations in Yeast Proliferating Cell Nuclear Antigen Define Distinct Sites for Interaction with DNA Polymerase δ and DNA Polymerase ε

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks

The lysosomal enzyme receptor protein (LERP) is not essential, but is implicated in lysosomal function in Drosophila melanogaster

The lysosomal enzyme receptor protein (LERP) of Drosophila melanogaster is the ortholog of the mammalian cation-independent mannose 6-phosphate (Man 6-P) receptor, which mediates trafficking of newly synthesized lysosomal acid hydrolases to lysosomes. However, flies lack the enzymes necessary to make the Man 6-P mark, and the amino acids implicated in Man 6-P binding by the mammalian receptor are not conserved in LERP. Thus, the function of LERP in sorting of lysosomal enzymes to lysosomes in Drosophila is unclear. Here, we analyze the consequence of LERP depletion in S2 cells and intact flies. RNAi-mediated knockdown of LERP in S2 cells had little or no effect on the cellular content or secretion of several lysosomal hydrolases. We generated a novel Lerp null mutation, LerpF6, which abolishes LERP protein expression. Lerp mutants have normal viability and fertility and display no overt phenotypes other than reduced body weight. Lerp mutant flies exhibit a 30–40% decrease in the level of several lysosomal hydrolases, and are hypersensitive to dietary chloroquine and starvation, consistent with impaired lysosome function. Loss of LERP also enhances an eye phenotype associated with defective autophagy. Our findings implicate Lerp in lysosome function and autophagy

Feeding \u3ci\u3eDrosophila\u3c/i\u3e a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns

Caloric restriction increases stress resistance and lifespan in Drosophila melanogaster and other species. The roles of individual nutrients in stress resistance and longevity are largely unknown. The vitamin biotin is a potential candidate for mediating these effects, given its known roles in stress signaling and gene regulation by epigenetic mechanisms, i.e., biotinylation of histones. Here, we tested the hypothesis that prolonged culture of Drosophila on biotin-deficient medium increases stress resistance and lifespan. Flies were fed a biotin-deficient diet for multiple generations; controls were fed a biotin-normal diet. In some experiments, a third group of flies was fed a biotin-deficient diet for 12 generations and then switched to control diets for two generations to eliminate potential effects of short-term biotin deficiency. Flies fed a biotin-deficient diet exhibited a 30% increase in lifespan. This increase was associated with enhanced resistance to the DNA-damaging agent hydroxyurea and heat stress. Also, fertility increased significantly compared with biotin-normal controls. Biotinylation of histones was barely detectable in biotin-deprived flies, suggesting that epigenetic events might have contributed to effects of biotin deprivation

Human SRCAP and Drosophila melanogaster DOM Are Homologs That Function in the Notch Signaling Pathway

The putative ATPase chromatin-remodeling machine SRCAP was identified in a yeast two-hybrid protein screen by interaction with the histone acetylase CBP. SRCAP is implicated in the transcriptional coactivation of cyclic AMP- and steroid-dependent promoters, but no natural chromosomal targets for SRCAP regulation have been identified. DOM is the unique SRCAP homolog in Drosophila melanogaster. The goal of this study was to test whether SRCAP is a functional homolog of DOM and to identify potential activities and targets of SRCAP in vivo. We show that human SRCAP complements recessive domino mutant phenotypes. This rescue depends on an intact ATPase homology domain. SRCAP colocalizes extensively with DOM on Drosophila polytene chromosomes and is recruited to sites of active transcription, such as steroid-regulated loci, but not to activated heat shock loci. We show that SRCAP recruits Drosophila CBP to ectopic chromosomal sites, providing the first evidence to suggest that SRCAP and CBP interact directly or indirectly on chromosomes. We show that DOM is a Notch pathway activator in Drosophila and that wild-type SRCAP—but not an ATPase domain mutant—can substitute for DOM in Notch-dependent wing development. We show that SRCAP potentiates Notch-dependent gene activation in HeLa cells. Taken together, these data implicate SRCAP and DOM in developmental gene activation

The unique GGA clathrin adaptor of Drosophila melanogaster is not essential.

The Golgi-localized, γ-ear-containing, ARF binding proteins (GGAs) are a highly conserved family of monomeric clathrin adaptor proteins implicated in clathrin-mediated protein sorting between the trans-Golgi network and endosomes. GGA RNAi knockdowns in Drosophila have resulted in conflicting data concerning whether the Drosophila GGA (dGGA) is essential. The goal of this study was to define the null phenotype for the unique Drosophila GGA. We describe two independently derived dGGA mutations. Neither allele expresses detectable dGGA protein. Homozygous and hemizygous flies with each allele are viable and fertile. In contrast to a previous report using RNAi knockdown, GGA mutant flies show no evidence of age-dependent retinal degeneration or cathepsin missorting. Our results demonstrate that several of the previous RNAi knockdown phenotypes were the result of off-target effects. However, GGA null flies are hypersensitive to dietary chloroquine and to starvation, implicating GGA in lysosomal function and autophagy

Drosophila ELL is associated with actively elongating RNA polymerase II on transcriptionally active sites in vivo

Several factors have been biochemically characterized based on their ability to increase the overall rate of transcription elongation catalyzed by the multiprotein complex RNA polymerase II (Pol II). Among these, the ELL family of elongation factors has been shown to increase the catalytic rate of transcription elongation in vitro by suppressing transient pausing. Several fundamental biological aspects of this class of elongation factors are not known. We have cloned the Drosophila homolog (dELL) in order to test whether ELL family proteins are actually associated with the elongating Pol II in vivo. Here we report that dELL is a nuclear protein, which, like its mammalian homologs, can increase the catalytic rate of transcription elongation by Pol II in vitro. Interestingly, we find that dELL co-localizes extensively with the phosphorylated, actively elongating form of Pol II at transcriptionally active sites on Drosophila polytene chromosomes. Furthermore, dELL is relocalized from a widespread distribution pattern on polytenes under normal conditions to very few transcriptionally active puff sites upon heat shock. This observation indicates a dynamic pattern of localization of dELL in cells, which is a predicted characteristic of a Pol II general elongation factor. We also demonstrate that dELL physically interacts with Pol II. Our results strongly suggest that dELL functions with elongating RNA polymerase II in vivo