Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control.

The mechanisms controlling duplication of the metazoan genome are only beginning to be understood. It is still unclear what organization of DNA sequences constitutes a chromosomal origin of DNA replication, and the regulation of origin activity during the cell cycle has not been fully revealed. We review recent results that indicate that chorion gene amplification in follicle cells of the Drosophila ovary is a model for investigating metazoan replication. Evaluation of cis sequence organization and function suggests that chorion loci share attributes with other replicons and provides insights into metazoan origin structure. Moreover, recent results indicate that chorion origins respond to S-phase control, but escape mechanisms that inhibit other origins from firing more than once in a cell cycle. Several identified genes that mediate amplification are critical for the cell cycle control of replication initiation. It is likely that further genetic screens for mutations that disrupt amplification will identify the cadre of proteins associated with origins and the regulatory pathways that control their activity. Furthermore, the recent development of methods to detect amplification in situ has uncovered new aspects of its developmental control. Examining this control will reveal links between developmental pathways and the cell cycle machinery. Visualization of amplifying chorion genes with high resolution also represents an opportunity to evaluate the influence of nuclear and chromosome structure on origin activity. The study of chorion amplification in Drosophila, therefore, provides great potential for the genetic and molecular dissection of metazoan replication.     

Molecular Evolution of Drosophila cdc6, an Essential DNA Replication-Licensing Gene,Suggests an Adaptive Choice of Replication Origins

Abstract Increased size of eukaryotic genomes necessitated the use of multiple origins of DNA replication, and presumably selected for their efficient spacing to ensure rapid DNA replication. The sequence of these origins remains undetermined in metazoan genomes, leaving important questions about the selective constraints acting on replication origins unanswered. We have chosen to study the evolution of proteins that recognize and define these origins every cell cycle, as a surrogate to the direct analysis of replication origins. Among these DNA replication proteins is the essential Cdc6 protein, which acts to license origins for replication. We find that two different species pairs of Drosophila show evidence of positive selection in Cdc6 in their highly conserved C-terminal AAA-ATPase domain. We also identified amino acid segments that are highly conserved in the N-terminal tail of Cdc6 proteins from various Drosophila species, but are not conserved even in closely related insect species. Instead, we find that the N-terminal tails of Cdc6 proteins vary extensively in size and sequence across different eukaryotic lineages. Our results suggest that choice of origin firing may be significantly altered in closely related species, as each set of replication proteins optimizes to its own genomic landscape.     

Molecular evolution of Drosophila Cdc6, an essential DNA replication-licensing gene, suggests an adaptive choice of replication origins

Increased size of eukaryotic genomes necessitated the use of multiple origins of DNA replication, and presumably selected for their efficient spacing to ensure rapid DNA replication. The sequence of these origins remains undetermined in metazoan genomes, leaving important questions about the selective constraints acting on replication origins unanswered. We have chosen to study the evolution of proteins that recognize and define these origins every cell cycle, as a surrogate to the direct analysis of replication origins. Among these DNA replication proteins is the essential Cdc6 protein, which acts to license origins for replication. We find that two different species pairs of Drosophila show evidence of positive selection in Cdc6 in their highly conserved C-terminal AAA-ATPase domain. We also identified amino acid segments that are highly conserved in the N-terminal tail of Cdc6 proteins from various Drosophila species, but are not conserved even in closely related insect species. Instead, we find that the N-terminal tails of Cdc6 proteins vary extensively in size and sequence across different eukaryotic lineages. Our results suggest that choice of origin firing may be significantly altered in closely related species, as each set of replication proteins optimizes to its own genomic landscape.     

Drosophila minichromosome maintenance 6 is required for chorion gene amplification and genomic replication

Duplication of the eukaryotic genome initiates from multiple origins of DNA replication whose activity is coordinated with the cell cycle. We have been studying the origins of DNA replication that control amplification of eggshell (chorion) genes during Drosophila oogenesis. Mutation of genes required for amplification results in a thin eggshell phenotype, allowing a genetic dissection of origin regulation. Herein, we show that one mutation corresponds to a subunit of the minichromosome maintenance (MCM) complex of proteins, MCM6. The binding of the MCM complex to origins in G1 as part of a prereplicative complex is critical for the cell cycle regulation of origin licensing. We find that MCM6 associates with other MCM subunits during amplification. These results suggest that chorion origins are bound by an amplification complex that contains MCM proteins and therefore resembles the prereplicative complex. Lethal alleles of MCM6 reveal it is essential for mitotic cycles and endocycles, and suggest that its function is mediated by ATP. We discuss the implications of these findings for the role of MCMs in the coordination of DNA replication during the cell cycle.     

Analysis of model replication origins in Drosophila reveals new aspects of the chromatin landscape and its relationship to origin activity and the prereplicative complex

Epigenetic regulation exerts a major influence on origins of DNA replication during development. The mechanisms for this regulation, however, are poorly defined. We showed previously that acetylation of nucleosomes regulates the origins that mediate developmental gene amplification during Drosophila oogenesis. Here we show that developmental activation of these origins is associated with acetylation of multiple histone lysines. Although these modifications are not unique to origin loci, we find that the level of acetylation is higher at the active origins and quantitatively correlated with the number of times these origins initiate replication. All of these acetylation marks were developmentally dynamic, rapidly increasing with origin activation and rapidly declining when the origins shut off and neighboring promoters turn on. Fine-scale analysis of the origins revealed that both hyperacetylation of nucleosomes and binding of the origin recognition complex (ORC) occur in a broad domain and that acetylation is highest on nucleosomes adjacent to one side of the major site of replication initiation. It was surprising to find that acetylation of some lysines depends on binding of ORC to the origin, suggesting that multiple histone acetyltransferases may be recruited during origin licensing. Our results reveal new insights into the origin epigenetic landscape and lead us to propose a chromatin switch model to explain the coordination of origin and promoter activity during development.     

The nuclear location and chromatin organization of active chorion amplification origins

It remains unclear how certain regions on metazoan chromosomes are selected to initiate DNA replication. In recent years a number of origins of DNA replication have been mapped, but there is still no DNA consensus for predicting where replication will initiate. Evidence suggests that the higher order structure of the nucleus and chromosome influences origin activity. Chromosomal DNA replication is proposed to occur in special compartments in the nucleus called replication foci. Foci in different regions of the nucleus initiate replication at different times of S-phase, suggesting nuclear position may contribute to where and when replication begins. Here we test the contribution of nuclear compartments for well-defined origins, those involved in amplification of the chorion (eggshell) genes during Drosophila oogenesis. The results of three-dimensional confocal microscopy indicate that chorion DNA replication origins are highly active in diverse positions within the nucleus. We also find that chorion replication origins inserted at ectopic chromosomal sites can amplify highly in diverse nuclear locations distinct from the endogenous loci, including when they are buffered against genomic position effects. We used fluorescence in situ hybridization to analyze chromosome structure during amplification. Contrary to the replication factory model, we find no evidence for spooling of DNA toward a replication center. We discuss the implications of these results for understanding the role of higher order structure in amplification and chromosome duplication.     

An orc1 allele with a mutated APC motif is female sterile with amplification defects

The origin recognition complex 1 (ORC1) is the largest subunit of the ORC, the heteromeric hexamer. ORC1 is an essential component of the pre-replicative complex (pre-RC) that licenses eukaryote DNA replication origins. The levels of ORC1 fluctuate during the mitotic cell cycle in Drosophila as well as in some human cells. Proteolysis of ORC1 occurs at the end of M phase in Drosophila, which is mediated by the anaphase-promoting complex (APC), and in late S phase in human cells by Skip-Cullin-F box (SCF). Previously we showed that proteolysis of ORC1 by APC is mediated by the ORC1 destruction box (the O-box), an APC motif conserved among species yet distinct from the D-box or KEN-box. Recently we showed that replacing the O-box with the D-box (ORC1^O→D) changes the degradation profile of ORC1 during a canonical cell cycle. Here we report further characterization of the ORC1^O→D allele that turned out to be a useful tool to examine the function of ORC1 in other modes of DNA replication during oogenesis. In endoreplication stages ORC1^O→D does not change any DNA content profiles, consistent with our previous finding that ORC is dispensable for endoreplication. However, in amplification stage replication efficiency of ORC1^O→D is drastically reduced, which resulted in amplification defects that led to thin egg shell phenotype. Taken together, our analyses show that orc1 allele newly identified is female sterile and possesses a unique feature of phenotypes that are distinct in different modes of DNA replication.     

Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2

It is important that chromosomes are duplicated only once per cell cycle. Over-replication is prevented by multiple mechanisms that block the reformation of a pre-replicative complex (pre-RC) onto origins in S and G2 phase. We have investigated the developmental regulation of Double-parked (Dup) protein, the Drosophila ortholog of Cdt1, a conserved and essential pre-RC component found in human and other organisms. We find that phosphorylation and degradation of Dup protein at G1/S requires cyclin E/CDK2. The N terminus of Dup, which contains ten potential CDK phosphorylation sites, is necessary and sufficient for Dup degradation during S phase of mitotic cycles and endocycles. Mutation of these ten phosphorylation sites, however, only partially stabilizes the protein, suggesting that multiple mechanisms ensure Dup degradation. This regulation is important because increased Dup protein is sufficient to induce profound rereplication and death of developing cells. Mis-expression has different effects on genomic replication than on developmental amplification from chorion origins. The C terminus alone has no effect on genomic replication, but it is better than full-length protein at stimulating amplification. Mutation of the Dup CDK sites increases genomic re-replication, but is dominant negative for amplification. These two results suggest that phosphorylation regulates Dup activity differently during these developmentally specific types of DNA replication. Moreover, the ability of the CDK site mutant to rapidly inhibit BrdU incorporation suggests that Dup is required for fork elongation during amplification. In the context of findings from human and other cells, our results indicate that stringent regulation of Dup protein is critical to protect genome integrity.     

The regulation of competence to replicate in meiosis by Cdc6 is conserved during evolution

DNA replication licensing is an important step in the cell cycle at which cells become competent for DNA replication. When the cell cycle is arrested for long periods of time, this competence is lost. This is the case for somatic cells arrested in G0 or vertebrate oocytes arrested in G2. CDC6 is a factor involved in replication initiation competence which is necessary for the recruitment of the MCM helicase complex to DNA replication origins. In Xenopus, we have previously shown that CDC6 is the only missing replication factor in the oocyte whose translation during meiotic maturation is necessary and sufficient to confer DNA replication competence to the egg before fertilization (Lemaitre et al., 2002: Mol Biol Cell 13:435-444; Whitmire et al., 2002: Nature 419:722-725). Here, we report that this oogenesis control has been acquired by metazoans during evolution and conserved up to mammals. We also show that, contrary to eukaryotic metazoans, in S. pombe cdc18 (the S. pombe CDC6 homologue), CDC6 protein synthesis is down regulated during meiosis. As such, the lack of cdc18 prevents DNA replication from occurring in spores, whereas the presence of cdc6 makes eggs competent for DNA replication.     

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. We demonstrate a role for the p21(CIP)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. Our data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts.     

Drosophila dMyc is required for ovary cell growth and endoreplication

Although the Myc oncogene has long been known to play a role in many human cancers, the mechanisms that mediate its effects in both normal cells and cancer cells are not fully understood. We have initiated a genetic analysis of the Drosophila homolog of the Myc oncoprotein (dMyc), which is encoded by the dm locus. We carried out mosaic analysis to elucidate the functions of dMyc in the germline and somatic cells of the ovary during oogenesis, a process that involves cell proliferation, differentiation and growth. Germline and somatic follicle cells mutant for dm exhibit a profound decrease in their ability to grow and to carry out endoreplication, a modified cell cycle in which DNA replication occurs in the absence of cell division. In contrast to its dramatic effects on growth and endoreplication, dMyc is dispensable for the mitotic division cycles of both germline and somatic components of the ovary. Surprisingly, despite their impaired ability to endoreplicate, dm mutant follicle cells appeared to carry out chorion gene amplification normally. Furthermore, in germline cysts in which the dm mutant cells comprised only a subset of the 16-cell cluster, we observed strictly cell-autonomous growth defects. However, in cases in which the entire germline cyst or the whole follicular epithelium was mutant for dm, the growth of the entire follicle, including the wild-type cells, was delayed. This observation indicates the existence of a signaling mechanism that acts to coordinate the growth rates of the germline and somatic components of the follicle. In summary, dMyc plays an essential role in promoting the rapid growth that must occur in both the germline and the surrounding follicle cells for oogenesis to proceed.     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.     

Schizosaccharomyces pombe Noc3 is essential for ribosome biogenesis and cell division but not DNA replication

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.     

Causal relations among cell cycle processes in Tetrahymena pyriformis. An analysis employing temperature-sensitive mutants

Utilization of temperature-sensitive mutants of Tetrahymena pyriformis affected in cell division or developmental pathway selection has permitted elucidation of causal dependencies interrelating micronuclear and macronuclear replication and division, oral development, and cytokinesis. In those mutants in which cell division is specifically blocked at restrictive temperatures, micronuclear division proceeds with somewhat accelerated periodicity but maintains normal coupling to predivision oral development. Macronuclear division is almost totally suppressed in an early acting mutant (mola) that prevents formation of the fission zone, and is variably affected in other mutants (such as mo3) that allow the fission zone to form but arrest constriction. However, macronuclear DNA synthesis can proceed for about four cycles in the nondividing mutant cells. A second class of mutants (psm) undergoes a switch of developmental pathway such that cells fail to enter division but instead repeatedly carry out an unusual type of oral replacement while growing in nutrient medium at the restrictive temperature. Under these circumstances no nuclei divide, yet macronuclear DNA accumulation continues. These results suggest that (a) macronuclear division is stringently affected by restriction of cell division, (b) micronuclear division and replication can continue in cells that are undergoing the type of oral development that is characteristic of division cycles, and (c) macronuclear DNA synthesis can continue in growing cells regardless of their developmental status. The observed relationships among events are consistent with the further suggestion that the cell cycle in this organism may consist of separate clusters of events. with a varying degree of coupling among clusters. A minimal model of the Tetrahymena cell cycle that takes these phenomena into account is suggested.     

An increase in mitochondrial DNA promotes nuclear DNA replication in yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD⁺-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.