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.     

Ectopic expression of the Suppressor of Underreplication gene inhibits endocycles but not the mitotic cell cycle in Drosophila melanogaster

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.     

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.     

Localization of a cis-acting element responsible for the developmentally regulated amplification of Drosophila chorion genes

Late in oogenesis two clusters of Drosophila chorion genes and flanking DNA sequences undergo specific amplification in ovarian follicle cells. Lines were constructed using P-element-mediated transformation in which DNA segments derived from the chorion gene cluster at 66D on chromosome III had been inserted at new chromosomal locations. Only transposons that contained a specific 3.8 kb genomic segment derived from the cluster underwent amplification during oogenesis, which occurred with apparently normal tissue and temporal specificity. Adjacent nonchorion sequences also underwent amplification. However, the ability of a transposon to replicate differentially was subject to position effect. These studies provide evidence for the existence of a specific, cis-acting element controlling chorion gene amplification, which includes an origin for disproportionate DNA replication. Attempts to induce amplification with subfragments of the 3.8 kb segment were unsuccessful, suggesting that much of this fragment may be required for amplification.     

Ectopic expression of the<Emphasis Type="Italic"> Suppressor of Underreplication</Emphasis> gene inhibits endocycles but not the mitotic cell cycle in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.Communicated by G. P. Georgiev     

Multiple replication origins are used during Drosophila chorion gene amplification

DNA from Drosophila egg chambers undergoing chorion gene amplification was analyzed using the two-dimensional gel technique of Brewer and Fangman. At stage 10, 34% of DNA molecules from the maximally amplified region of the third chromosome chorion gene cluster contained replication forks or bubbles. These nonlinear forms were intermediates in the process of amplification; they were confined to follicle cells, and were found only within the replicating region during the time of amplification. Multiple origins gave rise to these intermediates, since three separate regions of the third chromosome chorion locus contained replication bubbles. However, initiation was nonrandom; the majority of initiations appeared to occur near the Bgl II site located between the s18 and s15 chorion genes. The P[S6.9] chorion transposon also contained abundant replication intermediates in follicle cells from a transformed line. Initiation within P[S6.9] occurred near two previously defined cis-regulatory elements, one near the same Bgl II site (in the AER-d region) and one near the ACE3 element.     

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC.     

Characterization of a Drosophila Ortholog of the Cdc7 Kinase: A ROLE FOR Cdc7 IN ENDOREPLICATION INDEPENDENT OF CHIFFON*

Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G₁/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.     

DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding

Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction. DmORC displays at best six-fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA-binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30-fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator-initiator interactions and that in vivo other proteins must determine ORC's localization.     

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2–ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2–ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244–511), which competitively inhibited the TRF2–ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.     

Amplification enhancers and replication origins in the autosomal chorion gene cluster of Drosophila.

Drosophila melanogaster follicle cells over-replicate the chromosomal domain containing the third chromosome chorion gene cluster. Multiple regions of this cluster are needed in cis for attainment of high levels of amplification. We have confirmed the importance of the proposed amplification control element (ACE3) and demonstrated that it can support low levels of follicular amplification in the absence of other elements, but that it lacks detectable activity as a DNA replication origin. We have also demonstrated the existence of additional amplification-enhancing regions (AERs), by analyzing the amplification levels of a series of in situ induced, nested deletions of the chorion cluster. These deletions were induced by P-transposase perturbation of a chorion transposon in a highly amplifying transformed line, and were not accompanied by re-transposition, making possible a quantitative analysis of amplification levels in the absence of chromosomal position effects. Analysis of endogenous replication intermediates in wild-type follicular DNA suggested that at least one of the AERs may be an origin of replication and that amplification uses at least one additional replication origin.     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

Origin recognition complex binding to a metazoan replication origin

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.     

Developmentally regulated histone modifications in <Emphasis Type="Italic">Drosophila</Emphasis> follicle cells: initiation of gene amplification is associated with histone H3 and H4 hyperacetylation and H1 phosphorylation

We have used gene amplification in Drosophila follicle cells as a model of metazoan DNA replication to address whether changes in histone modifications are associated with replication origin activation. We observe that replication initiation is associated with distinct histone modifications. Acetylated lysines K5, K8, and K12 on histone H4 and K14 on histone H3 are specifically enriched during replication initiation at the amplification origins. Strikingly, H4 acetylation persists at an amplification origin well after replication forks have progressed significantly outward from the origin, indicating that H4 acetylation is associated with origin regulation and not histone deposition at the replication forks. Origin recognition complex subunit 2 (orc2) mutants with severe amplification defects do not abolish H4 acetylation, whereas the dup/cdt1 mutant delays the appearance of acetylation foci, and mutants in rbf result in temporal persistence. These data indicate that core histone acetylation is associated with origin activity. Furthermore, follicle cells undergoing gene amplification exhibit high levels of histone H1 phosphorylation. The patterns of H1 phosphorylation provide insights into cell cycle states during amplification, as H1 kinase activity in follicle cells is responsive to high Cyclin E activity, and it can be abolished by overexpressing the retinoblastoma homolog, Rbf, that represses Cyclin E. These data suggest that amplification origins are able to initiate when the cells are in a late S-phase, when the genome is normally not licensed for replication.