Functional Analysis of an Orc6 Mutant in Drosophila

Functional analysis of an Orc6 mutant in Drosophila

Maxim Balasov, Richard P. H. Huijbregts, and Igor Chesnokov1

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham School of Medicine, 720 20th Street South, Birmingham, AL 35294

Edited by Michael R. Botchan, University of California, Berkeley, CA, and approved April 22, 2009 (received for review March 12, 2009) The origin recognition complex (ORC) is a 6-subunit complex have a function in cytokinesis (7, 22, 23). This function in required for the initiation of DNA replication in eukaryotic organ- Drosophila is attributed to the C-terminal domain of Orc6 (22). isms. ORC is also involved in other cell functions. The smallest To study the Orc6 functions in a living organism, we generated Drosophila ORC subunit, Orc6, is important for both DNA replica- and characterized the Orc6-deletion mutant in Drosophila.We tion and cytokinesis. To study the role of Orc6 in vivo, the orc6 gene have analyzed in detail the mutant phenotypes associated with was deleted by imprecise excision of P element. Lethal alleles of a lethal allele of the Drosophila orc6 gene alone or with different orc6 are defective in DNA replication and also show abnormal versions of fly or human Orc6 rescue transgenes, gaining further chromosome condensation and segregation. The analysis of cells insight into the roles Orc6 plays through the cell cycle in containing the orc6 deletion revealed that they arrest in both the metazoan species. G1 and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of Results mutant transgenes of Orc6 with deleted or mutated C-terminal Drosophila Orc6 Accumulates on Chromosomes in Late Mitosis. In domain results in a release of mutant cells from G1 arrest and Drosophila cells Orc6 colocalizes with other ORC subunits but restoration of DNA replication, indicating that the DNA replication also displays distinct cytoplasmic and membrane staining in both function of Orc6 is associated with its N-terminal domain. How- embryonic and tissue culture cells, reflecting its functions in both ever, these mutant cells accumulate at mitosis, suggesting that the DNA replication and cytokinesis (17, 22). Analysis of mitotic C-terminal domain of Orc6 is important for the passage through stages in developing Drosophila neuroblasts revealed that at the M phase. In a cross-species complementation experiment, the prometaphase and metaphase Orc6 was present in the nucleus

expression of human Orc6 in Drosophila Orc6 mutant cells rescued but was weakly associated with the DNA (Fig. 1). However, CELL BIOLOGY DNA replication, suggesting that this function of the protein is beginning at anaphase, Orc6 staining of the segregating chro- conserved among metazoans. mosomes became intense along the length of the chromatids and persisted further into telophase (Fig. 1). The observed pattern of ͉ ͉ DNA replication ORC Chromatin Orc6 staining in this experiment is remarkably similar to those of both Drosophila Orc2 and Xenopus Orc1, which were also he hexameric origin recognition complex (ORC) is an es- weakly associated with DNA at metaphase but present at the Tsential component for eukaryotic DNA replication. It was later stages of mitosis (4, 24, 25). Most likely, at these stages originally discovered in Saccharomyces cerevisiae, and subse- ORC is deposited onto the replication origins in preparation for quent studies both in yeast and in higher eukaryotes laid the the next cell cycle. foundation for understanding the functions of this important key initiation factor. ORC binds to origin sites in an ATP-dependent Generation of an Orc6 Mutation in Drosophila. To study the func- manner and directs the assembly of the prereplicative complex tions of Orc6 in vivo in live animals, we generated a deletion of (pre-RC) at the origins (1, 2). ORC subunits and/or complete the orc6 gene in Drosophila by using the method of P element ORC complexes have also been identified in many metazoan imprecise excision. Several lethal deletions of the orc6 genomic species (1, 3), suggesting the existence of common mechanisms region were identified and their boundaries mapped by sequenc- for the initiation of DNA replication in all eukaryotes. ORC ing. Fig. 2A shows a map of the genomic region of the second genes are essential for cell survival. Mutational analysis of chromosome containing the orc6 gene, and it also shows the ORC-related genes in yeast and in higher eukaryotes reveals boundaries of the obtained deletion used in the current study. defects in DNA replication (reviewed in refs. 1 and 3). In other This third-instar lethal deletion, called orc635, includes the whole studies, immunodepletion experiments using either Xenopus or orc6 gene and a part of overlapping CG1667, which has no Drosophila replication-competent extracts indicate an absolute apparent or predicted function. requirement for ORC to initiate DNA replication (4–6). Acute To rescue the orc635 deletion, we used a 3.3-kb genomic clone depletion of ORC gene expression in human cells by RNAi containing the wild-type orc6 gene together with whole CG1667. resulted in cell cycle arrest (7, 8). In addition to initiating DNA This genomic construct is depicted in Fig. 2A. In addition, replication, ORC is involved in other functions described pre- full-length, GFP-fused Orc6 transgene under control of the viously in detail (1, 3, 9). native Orc6 promoter was used to rescue the lethality associated The Orc6 protein is the least conserved of all ORC subunits. with the obtained deletion. Both constructs, the genomic clone In S. cerevisiae, Orc6 is not important for DNA binding, but it is containing the orc6 gene and CG1667, as well as the full-length required for cell survival (10–12) and the maintenance of the GFP-Orc6 transgene alone—were successfully able to rescue the pre-RC, specifically for recruitment of Cdt1 followed by MCM deletion mutant (Fig. 2B), demonstrating that the lethality is loading (13, 14). Schizosaccharomyces pombe and metazoan Orc6 proteins (6, 15, 16) are more homologous, similar in size, and considerably smaller than the S. cerevisiae Orc6. In Dro- Author contributions: M.B. and I.C. designed research; M.B., R.P.H.H., and I.C. performed sophila, Orc6 is essential for ORC-dependent DNA binding and research; R.P.H.H. contributed new reagents/analytic tools; M.B. and I.C. analyzed data; DNA replication (17, 18). In Xenopus and human systems Orc6 and I.C. wrote the paper. is less tightly associated with the core complex, and some of the The authors declare no conﬂict of interest. published data suggest that Orc6 may not be important for these This article is a PNAS Direct Submission. activities (19–21). This apparent inconsistency may reflect the 1To whom correspondence should be addressed. E-mail: [email protected]. difference in affinity of Orc6 for the core ORC1-5 complex in This article contains supporting information online at www.pnas.org/cgi/content/full/ distant metazoan species. Drosophila Orc6 and human Orc6 also 0902670106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902670106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 Prometaphase Metaphase Anaphase A Anaphase B Telophase DAPI Orc6 Merge

Fig. 1. Drosophila Orc6 accumulates on chromosomes in anaphase through telophase. Immunofluorescence images of wild-type Drosophila neuroblasts stained with affinity-purified anti-Orc6 antibody (green) are shown in metaphase, anaphase, and telophase stages. DNA is stained with DAPI (blue). (Scale bar: 5 ␮m.)

indeed associated with the deleted orc6 gene. CG1667 had no effect on viability in these experiments. Next, we tested the ability of the human orc6 gene to rescue the orc635 deletion. As shown in Fig. 2B, the expression of the full-length GFP-fused human Orc6 protein in Drosophila orc635 did not rescue lethal orc635 mutation, indicating that these 2 protein homologues Fig. 2. Generation and rescue of Orc6 mutant. Fragment of genomic map cannot substitute each other in viability experiments. 35 Orc6 protein consists of 2 functional domains, which are from Drosophila database and limits of the orc6 deletion are shown (A). Drosophila wild-type (DmOrc6), human (HsOrc6), truncated C terminus mu- important for DNA replication and cytokinesis functions (22). tants (DmOrc6-220, DmOrc6-200), and substitution mutant (DmOrc6- The larger, N-terminal domain is important for replication, WK228AA), all fused with GFP, were used in the rescue experiments (B). The whereas the smaller, C-terminal domain is required for the predicted cytokinesis domain of Orc6 is shown in white. Conservative trypto- interaction with Pnut protein and cytokinesis function of Orc6, phan (228) and lysine (229) amino acid residues of Drosophila Orc6 were as demonstrated by our earlier in vitro and cell culture-based replaced with alanines to create DmOrc6-WK228AA clone. An alignment of studies (18, 23). To learn more about the functions of the corresponding Orc6 sequences between different species is shown in the box. C-terminal domain in vivo, we designed GFP-Orc6 transgenes containing C-terminal deletions or point mutations and used animals, indicating that brain development and cell proliferation them in rescue experiments. Two C-terminal deletion mutations, Orc6-200 and Orc6-220, were used. These mutations do not stop prematurely when the maternal supply of Orc6 protein is interfere with the replicative function of Orc6 and reconstituted depleted. BrdU incorporation was also severely reduced in ORC in vitro; however, they are defective for the interaction homozygous Orc6-deletion mutant larval brains compared with with Pnut protein and induce cytokinetic defects when expressed the large levels of BrdU incorporation in heterozygous brains in Drosophila tissue culture cells (22). The Orc6-WK228AA (Fig. 3A), demonstrating the lack of DNA replication. Corre- point amino acid mutant was chosen based on sequence analysis spondingly, immunoblot analysis of Orc6-null mutant brain of Orc6 homologues derived from different species of animals extracts did not show the presence of Orc6 protein compared and plants. As shown in Fig. 2B, W228 and K229 are conserved with the immunoblots using heterozygous brain cell extracts (Fig. amino acids in metazoan and plant organisms and are localized S2). No imaginal discs were detected in late third instar larvae at the extreme C terminus of Orc6, just beyond the boundary of of mutant animals. These phenotypes represent severe prolifer- the Orc6-220 deletion. We hypothesized that these amino acids ation defects also observed in other replication initiation factor might be important for nonreplicative functions of Orc6. As mutants (24, 26–30). expected, no rescue to viability was observed when C-terminal Recently, it was reported that Orc1 was not required for Orc6 mutants were used (Fig. 2B), suggesting that this domain endoreplication in Drosophila salivary glands (30). We analyzed of Orc6 is important for survival of the animals. The detailed DNA replication in salivary glands isolated from Orc6 mutant analysis of the described rescue experiments with all Orc6 clones animals. Salivary glands appeared normal morphologically but is presented in Table S1. All GFP-Orc6 transgenes used in this were reduced in size. BrdU incorporation in salivary glands was study were tested for expression in Drosophila tissues by Western close to the levels of the wild-type or heterozygous animals (Fig. blot analysis (Fig. S1). All transgenes expressed in vivo at similar 3B), suggesting that DNA replication was not affected by the levels. deletion of Orc6. We have also tested the salivary glands of Orc6-deletion mutant animals for the presence of Orc6 protein

Orc6 Mutant Is Defective in DNA Replication and Arrests in both G1 and by immunostaining and Western blotting experiments. Immu- Mitosis. To investigate whether the Orc6 mutant obtained in the noblotting experiments did not reveal detectable amounts of course of our studies is also defective for DNA synthesis, BrdU Orc6 in the protein extracts isolated from the salivary glands of incorporation levels in larval neuroblasts were determined (Fig. mutant animals compared with heterozygous flies (Fig. S2). 3A). The optic lobes of Orc6 mutant brains were significantly However, immunostaining experiments using several indepen- smaller in size compared with the wild-type or heterozygous dently prepared batches of affinity-purified Orc6 antibody con-

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902670106 Balasov et al. Downloaded by guest on September 25, 2021 35 Table 1. Effect of Orc6 transgenes on G1 arrest of the orc6 neuroblasts No. of cells

G1, S/G2/M, G1:S/G2/M Fly genotype 1 centrosome 2 centrosomes ratio

orc635/Cy 154 119 1.3:1 orc635/orc635 208 28 7.4:1 orc635/Cy, GFP-orc6 286 250 1.1:1 orc635/orc635, GFP-orc6 234 211 1.1:1 orc635 /Cy, GFP-orc6-200 108 93 1.2:1 orc635/orc635, GFP-orc6-200 324 33 9.8:1 orc635/Cy, GFP-orc6-220 267 243 1.1:1 orc635/orc635, GFP-orc6-220 235 187 1.2:1 orc635/Cy, GFP-orc6-WK228AA 176 144 1.2:1 orc635/orc635,GFP-orc6-WK228AA 256 236 1.1:1 orc635/Cy, GFP-Hs-orc6 141 139 1.01:1 orc635/orc635, GFP-Hs-orc6 317 221 1.4:1

Several independent ﬂy stocks have been tested for each GFP-Orc6 transgene. In every case, tested stocks have shown similar G1 to S/G2/M ratios.

the wild-type or heterozygous tissues, probably because of a large number of cells arrested at G1 phase (Table 2, second row). Fig. 3. DNA replication in Orc6 mutant. BrdU incorporation level (red) in At the next step, chromosome spreads were scored for the third-instar larval brains (A) and in salivary glands of orc635 mutant (B) presence of chromosomes in each stage of mitosis. No cells at 35 compared with wild-type or orc6 /Cy heterozygous animals. (C) Immunoflu- anaphase or telophase stages were found in cells carrying the CELL BIOLOGY orescence experiments using affinity-purified anti-Orc6 antibody on polytene orc635 deletion. Instead, a large number of metaphase-like chromosomes isolated from wild-type animals (red) and from orc635 mutant figures were observed, in which highly condensed chromosomes (green) show the presence of Orc6 at specific loci along the chromosome were present with incomplete or disorganized alignment (Fig. 4). ␮ ␮ length and in the nucleolus (N). (Scale bars: A and B, 100 m; C,50 m.) In addition, individual chromatids appeared aberrantly condensed and fragmented. Chromosome spreads were prepared sistently revealed distinct Orc6-positive bands on salivary gland from third instar larval brains to analyze the degree of chromo- orc635 homozygous cells. The represen- chromosomes isolated from Orc6-null homozygous flies (Fig. some condensation in tative data from wild-type and mutant cells are shown in Fig. 4. 3C). The number of Orc6-positive bands was reduced, but In general, the mitotic chromosomes in mutant neuroblasts nevertheless always detectable on the polytene chromosomes appeared shorter and thicker, and sister chromatid cohesion was prepared from the salivary glands of Orc6 mutant flies. These frequently lost (Fig. 4). Extensive chromosome fragmentation results suggest that low amounts of maternally deposited Orc6 was also observed. Therefore, we conclude that Orc6-deletion protein are still present in salivary glands and may therefore mutant, in addition to the G1 arrest, also has a defect in contribute to the observed levels of DNA replication. establishing a true metaphase and progressing past this point. Our immunostaining experiments also revealed high amounts of Orc6 localized in the nucleolus of the salivary gland cells (Fig. Effect of Orc6 Transgenes on DNA Replication and Cell Cycle Progres- 3C). We have observed similar staining patterns for other sion in the Orc6-Deletion Mutant. Next, we analyzed the effect of Drosophila ORC subunits, including Orc1 and Orc2. Moreover, transgenes, described in Fig. 2, on DNA replication, cell cycle ORC was found in the nucleolus in human cells (31, 32). These progression, and cell morphology. As shown in Fig. 2B and Table findings may indicate a potential interaction of ORC with S1, only transgenes containing the full-length orc6 gene were components of the ribosome biogenesis pathway (31, 33). Earlier studies of Orc2 and Orc5 mutants in Drosophila confirmed that cells bearing these mutations arrest both in G1 Table 2. Effect of GFP-Orc6 transgenes on cell cycle progression and during mitosis (24, 29). We have also found that the Orc6 of orc635 mutant neuroblasts mutant has double arrest points during the cell cycle. First, Fly genotype Mitotic to total cells ratio (%) determination of the ratio of cells with single centrosomes to those with duplicated centrosomes clearly confirmed that Dro- orc635/Cy 79:3,677 (2.1) sophila Orc6-null cells were delayed in cell cycle progression orc635/orc635 18:1,425 (1.2) (Table 1). Only a single centrosome is apparent when neuro- orc635/Cy, GFP-orc6 45:2,384 (1.9) orc635/orc635, GFP-orc6 25:1,488 (1.7) blasts enter G1 phase. During S phase the centrosomes start orc635/Cy, GFP-orc6-200 64:2,495 (2.6) separating, and 2 centrosomes are apparent in G2 phase. Further, during mitosis, the 2 poles of the spindle are also clearly marked orc635/orc635, GFP-orc6-200 11:1,777 (0.6) by ␥-tubulin antibody staining. By using this assay, the ratio of orc635/Cy, GFP-orc6-220 23:1,712 (1.3) orc635/orc635,GFP- orc6-220 31:764 (4) G1 cells was found to be increased by a factor of Ϸ7inthe 35 Drosophila Orc6-deletion mutant, demonstrating the accumula- orc6 /Cy, GFP-orc6-WK228AA 16:802 (1.9) orc635/orc635, GFP-orc6-WK228AA 180:2,541 (7) tion of G1 cells in the absence of Orc6 protein (Table 1, second 35 row). Second, we identified mitotic cells by immunostaining of orc6 /Cy, GFP-Hs-orc6 60:3,200 (1.8) 35 35 Drosophila neuroblasts with antibody against the mitotic marker orc6 /orc6 , GFP-Hs-orc6 64:3,738 (1.7) phosphohistone H3. Overall, the number of mitotic cells ob- At least 3 independent fly stocks have been tested for each GFP-Orc6 served in the Orc6-deletion mutant decreased compared with transgene.

Balasov et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 25, 2021 wt metaphase orc635 metaphases A B C D

wt anaphase A’ B’ C’ E

Fig. 6. Cell cycle defects in Orc6 transgenic mutants. orc635 neuroblasts expressing GFP-Orc6-220 or GFP-Orc6-WK228AA are arrested in metaphase- 35 Fig. 4. Aceto-Orcein squashes of orc6 brains. Metaphase-like chromo- like stage (A), but they also display anaphase-like ﬁgures (B) and proceed 35 somes in orc6 mutant neuroblasts are abnormally condensed and frag- further in the cell cycle to form cells with multiple centrosomes and polyploid ␮ mented compared with the wild-type chromosomes (wt). (Scale bar: 5 m.) cells (C–E). Two clusters of multicentrosome bodies (enlarged 5ϫ in CЈ Insets) are shown on metaphase-like ﬁgures in C. DNA is stained with DAPI (blue); centrosomes are stained with anti-␥-tubulin antibody (red); mitotic chromo- 35 able to rescue orc6 flies to the adult stage, confirming that somes are stained with antibody raised against phosphohistone H3 (green). full-length Orc6 protein is required for viability. Transgenic Merged images are shown in AЈ, BЈ, CЈ, D, and E. (Scale bar: 10 ␮m.) orc635 animals carrying constructs containing either human orc6 gene or Drosophila Orc6 with deletions or point mutations within the C-terminal domain of the protein did not survive beyond no BrdU incorporation was detected in brain neuroblasts. How- third larval instar stage. However, detailed analysis revealed that ever, chromosomes from neuroblasts isolated from Orc6-null Orc6-null cells carrying these transgenes did not exhibit the G1 mutant carrying Orc6-220, Orc6-WK228AA, or human Orc6 arrest characteristic for all described ORC mutants in Drosoph- transgenes were all able to incorporate BrdU, and therefore were ila. Again, we quantified the ratio of cells containing 1 and 2 able to replicate DNA (Fig. 5). Brains observed in these larvae centrosomes for all transgenes used in this study. The results are were still significantly reduced in size compared with heterozy- summarized in Table 1. The number of cells in G1 stage is gous or wild-type larval brains, suggesting severe defects in cell increased by a factor of Ϸ7intheDrosophila orc635 mutant, proliferation and larval brain development. demonstrating the accumulation of G1 cells in the absence of Further analysis revealed that neuroblasts carrying the Orc6- Orc6 protein (Table 1, second row). However, the expression of 220 and Orc6-WK228AA transgenes accumulated at mitosis the Orc6-220-deletion mutant, Orc6-WK228AA mutant, or hu- (Table 2). The number of mitotic neuroblasts derived from these man Orc6 protein resulted in the release of orc635 mutant cells transgenic flies increased 4- to 8-fold compared with orc635 from G1 arrest (Table 1). In other words, the ratio of cells mutant cells. The expression of the shorter Orc6-200 transgene containing either 1 or 2 centrosomes was indistinguishable from did not have any effect, and the number of mitotic cells carrying cells obtained from heterozygous siblings for all of these trans- this transgene was indistinguishable from the orc635 homozygous genes, with the sole exception of Orc6-200, which did not differ mutant neuroblasts and consistently lower than in wild-type or from orc635 homozygous mutant. heterozygous tissues. The ratio of mitotic to total cells in orc635 Next, we asked whether these orc635 mutant cells containing neuroblasts expressing human Orc6 protein increased by the different Orc6 transgenic constructs would be able to pass factor of Ϸ2 compared with Orc6-null cells and was close to the through the S phase and replicate DNA. The results are present ratio of mitotic to total cells in heterozygous sibling brains. in Fig. 5. Again, the orc635 mutant carrying the Orc6-200 gene Again, a large number of metaphase-like figures were ob- did not differ in that respect from orc635 mutant cells, because served in cells expressing Orc6 C-terminal mutants (Fig. 6A), similarly to Orc6-null cells. However, we also observed cells at different mitotic stages, cells with multiple centrosomes, and 35 35 35 35 orc6 /orc6 orc6 /Cy orc6 , orc6-200 polyploid cells when Orc6-220 and Orc6-WK228AA transgenes ABC were expressed in orc635 mutant. For example, anaphase-like figures containing broken chromosomes were observed (Fig. 6B). These anaphase chromosomes contained mutant Orc6 proteins, as shown in Fig. S3 for Orc6-WK228AA protein. Polyploid cells with multiple centrosomes (Fig. 6 C and D) as well

orc635 , orc6-220 orc635 , orc6-WK228AA orc635 , Hs-orc6 as cells containing multiple centrosomes with almost no DNA (Fig. 6E) were also detected. DEF Discussion In our studies, we have analyzed in detail the mutant phenotype associated with a lethal allele of the Drosophila orc6 gene, gaining further insight into Orc6 functions during the cell cycle in metazoan species. The Orc6 protein is the most evolutionary Fig. 5. BrdU incorporation in Orc6 transgenic mutants. DNA replication in diverged and enigmatic among all ORC subunits because its orc635 mutant neuroblasts (A) is rescued by the expression of Drosophila GFP-Orc6-220 (C terminus-truncated mutant; D), GFP-Orc6-WK228AA (amino functions in various eukaryotic organisms differ. Orc6 is dis- acid substitution mutant; E), and human GFP-HsOrc6 (F), but not by the pensable for DNA binding in budding yeast (12) but is required expression of Drosophila GFP-Orc6-200 (C). BrdU incorporation in heterozy- for origin recognition in Drosophila (17, 18). Orc6 is tightly gous orc635/Cy neuroblasts is shown in B. (Scale bar: 100 ␮m.) associated with core ORC subunits in yeast and Drosophila, and

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902670106 Balasov et al. Downloaded by guest on September 25, 2021 it is crucial for DNA replication in these species (1, 6, 12). In organism. We found that only the constructs containing a contrast, human Orc6 is loosely bound to the core complex (20), full-length Drosophila Orc6 gene, but not its C-terminal mutants, and its role in DNA replication has not yet been completely were able to rescue orc6Ϫ animals to viability, indicating that established. This controversy has partially resulted from the use both domains of Orc6 are important for animal survival. C- of different experimental systems that generally do not permit an terminal mutants of Orc6 were active in replication and DNA organismal view of the DNA replication process under physio- binding in vitro and in cell culture assays (18, 22). Therefore, a logical conditions. priori we expected to observe at least partial rescue of DNA The functional ORC complex begins to assemble on chromo- replication in developing larval tissues expressing these mutants. somes during anaphase. We have found that during mitotic Indeed, constructs containing Drosophila Orc6-220 deletion stages, Orc6 localization is remarkably similar to that of other mutant and Orc6-WK228AA, and also human Orc6, were able ORC subunits, such as Drosophila Orc2 and Xenopus Orc1. All to rescue DNA replication in brain neuroblasts of orc635 larvae. of these proteins were weakly associated with DNA at metaphase Cells arrested previously at G1 stage were now able to pass G1 but present at the later stages of mitosis (4, 24, 25). This pattern arrest block and undergo a round of DNA replication. However, of binding of Drosophila ORC to the chromatin depends on the brains in orc635 animals containing these transgenes were still cessation of mitotic cyclin activity (25). Most likely, at these significantly smaller and undeveloped compared with the wild- stages functional 6-subunit ORC is deposited onto the replica- type or heterozygous flies. Imaginal discs were also not detected, tion origins in preparation for the next cell cycle. suggesting that normal cell cycle progression and tissue devel- The analysis of a mutant phenotype for the Orc6 gene in opment were not restored. Consistent with this observation, we Drosophila during the course of our studies confirmed in a living found a significant increase in neuroblasts arrested during animal the important role of Orc6 in cell functions. Similarly to mitosis, indicating that the N-terminal domain of Orc6 is not the other reported ORC mutants (24, 27–30), we observed vastly sufficient to rescue the mitotic function of the protein. It is also reduced BrdU labeling in the proliferative neural tissue of larvae interesting to note that the Orc6-200 mutant, which was active in homozygous for the Orc6 mutation, consistent with a crucial role DNA binding and replication in vitro, failed to rescue DNA for ORC in the DNA replication. These data together with the replication in our current in vivo study. This shorter Orc6-200 increased occurrence of cells with a single centrosome indicate protein was also less tightly associated with core ORC1-5 an early cell cycle delay, resulting in G1 arrest in Orc6-null subunits during reconstitution and purification assays, suggest- mutant neuroblasts. The amount of cells arrested in G1 increased ing that this particular deletion may extend deeper into the Ϸ7-fold compared with the wild-type or heterozygous cells, replicative domain of the protein, resulting in observed replica- CELL BIOLOGY suggesting that the inactivation of Orc6 leads to defects in tion defects in live animals. pre-RC assembly. Some of the orc635 mutant cells, however, Orc6 is the least conserved ORC subunit; however, human accumulated at a stage with many characteristic features of Orc6 protein was able to rescue DNA replication in Drosophila metaphase. Metaphase arrest with abnormally condensed chro- larval brains lacking the endogenous Orc6. Orc6 in human cells mosomes has also been observed in other ORC and replication is loosely bound to core ORC and also found to be dispensable protein mutants (24, 29). It appears that in all of these cases, for DNA binding and DNA replication on Orc2-depleted Xe- insufficient levels of replication proteins due to gradually de- nopus egg extracts (21, 34). Other studies have shown that Orc6 pleted maternal deposits result in incomplete DNA replication is important for DNA replication, because depletion of the and broken chromosomes that would lead to cell cycle arrest at protein from human cells results in DNA replication defects (7). the early stages of mitosis by a mechanism sensitive to chromo- In our current studies the expression of human Orc6 in Dro- Ϫ 35 somal integrity. sophila orc6 cells relieved an observed G1 arrest in orc6 Recent studies on an Orc1 mutant suggested that ORC may be mutants, resulting in a rescue of DNA replication. The ability of dispensable for the endoreplication in Drosophila (30). Our human Orc6 protein to support DNA replication in Drosophila analysis of the Orc6-deletion mutant similarly revealed that cells suggests that the 2 proteins are homologous in replication despite DNA replication defects observed in Drosophila brain function and also provides an opportunity for further molecular cells, the development and DNA synthesis in salivary glands were dissection of human Orc6 in vivo, by using Drosophila as a model similar to the wild-type or heterozygous cells. Western blotting system. experiments did not reveal detectable Orc6 protein in salivary Despite their ability to replicate DNA, the orc635 mutant cells glands isolated from homozygous mutant animals. It is possible carrying the Orc6-220, Orc6-WK228AA, and human Orc6 trans- that as maternal supplies of Orc6 are depleted, the Western genes accumulated at the stage of mitosis with the significant blotting analysis may not be sensitive enough to detect the increase in the number of mitotic cells, compared with orc6-null residual Orc6 protein. Immunofluorescence experiments, how- mutant. Many cells were arrested with metaphase-like figures ever, consistently showed Orc6-specific bands throughout poly- with uncondensed and broken chromosomes, suggesting that tene chromosomes at the same stage. The number of the DNA may be underreplicated in these mutants. Arrest at meta- Orc6-positive bands was Ϸ5- to 10-fold lower in polytene phase also suggests that C-terminal Orc6 mutants may be chromosomes derived from Orc6-deletion mutant cells than in defective during ORC-dependent assembly of pre-RC at the chromosomes from control wild-type or heterozygous cells, but final stages of mitosis. It is possible that the N terminus of Orc6 they were always detectable with many independent batches of is necessary, but not wholly sufficient, to rescue all Orc6 func- affinity-purified rabbit polyclonal or mouse monoclonal anti- tions in DNA replication. However, we also found cells at body against Drosophila Orc6. Thus, we cannot at this point different mitotic stages, which suggests that some cells escape exclude the possibility that the residual maternal deposits of metaphase arrest. Furthermore, the appearance of multinucle- Orc6 protein are sufficient to support endoreplication in the ated and polyploid cells as well as cells with multiple centrosomes salivary glands, augmented by a lower turnover of ORC in indicates that cell cycle progression through mitosis and cyto- polyploid cells than in mitotically active cells. kinesis is defective in cells carrying C-terminal mutations of Orc6 From our earlier biochemical and cell culture studies of Orc6 protein. The role of ORC subunits in coordinating DNA repli- activities, we concluded that Orc6 protein in Drosophila con- cation and centrosome copy number in human cells has been sisted of 2 functional domains important for DNA binding and reported recently (35). replication (18), as well as for cytokinesis, through interaction Overall, our data provide evidence in a living organism that with the septin protein Pnut (22, 23). In the current study, we DNA replication function of Orc6 is associated mainly with its wanted to address the functions of Drosophila Orc6 in a living N-terminal domain, whereas the C-terminal domain is necessary

Balasov et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 25, 2021 for the passage through the M phase. The whole Orc6 protein, clone was created by PCR of wild-type genomic DNA with primers 5Ј- however, is required for the survival of Drosophila. DNA rep- CCACAAATATGGCACGATTGC-3Ј and 5Ј-CGAAGACAGCGACAATGAGACG-3Ј. lication function of Orc6 can also be rescued by human Orc6 The PCR product was double-digested with EcoRI/XbaI and cloned into the protein, suggesting the conservation of replicative function pUAS vector. Obtained constructs were sequenced, purified, and injected into w1118 fly embryos. The expression of fused products was verified by Western among metazoan species. blot analysis with anti-Orc6 antibody. In rescue experiments of the orc6 mutant, progeny from heterozygous orc6/Cy; GFP-Orc6/ϩ were analyzed for Materials and Methods the presence of orc6/orc6; GFP-Orc6 flies. The percentage of rescued flies was Generation of Orc6-Deletion Mutant. Deletions of Orc6 gene have been gen- calculated based on expected segregation. erated by imperfect excision of P element-based transposon P{EPgy2}EY04071 (FBti0027288). This transposon is mapped 71 bp upstream of the Orc6 start Site-Directed Mutagenesis. The 2-amino acid mutant Orc6-WK228AA was codon, according to the sequence information released by the Gene Disrup- generated by replacing amino acids W228 and K229 with alanines following tion Project. To initiate excision, the homozygous females y1w67c23; the Stratagene site-directed mutagenesis protocol (www.stratagene.com/ ϩ ϩ P{w mCy mDint2ϭEPgy2}Ey04071 (Bloomington stock 15714) were crossed to manual/200516.pdf). males of jump stock y1w1118; CyO, PBac{wϩmCϭDelta 2-3. Exel}2/amosTft, bearing transposase on a second chromosome, marked by Curly.F1 Curly male Third-Instar Larval Brain Squashes. Brains of third-instar larvae were squashed 1 67c23 ϩmC ϩmDint2ϭ ϩmCϭ progeny y w ; P{w y EPgy2}Ey04071/CyO, PBac{w Delta in acetoorcein without colchicine treatment and hypotonic shock, following 1 1118 2-3. Exel}2 were collected and crossed to y w ; If/CyO females. The resulting a general protocol described previously (37). This procedure allows observa- F2 progeny were screened for white-eyed flies. White-eyed flies were crossed tion of all phases of mitosis. individually to y1w1118; If /CyO to set up stocks. Genomic DNA of these mutants Ј was isolated and analyzed by PCR with primers mmup7: 5 -GAGTACCTCGAC- BrdU Labeling and Immunostaining of Third-Instar Larval Brains. Larval brains Ј Ј Ј CATGTTGCC-3 and mmdown1: 5 -AATGCCACGACGCTGTTTCTG-3 or were soaked in PBS with 1 ␮M BrdU for 30 min at 25 °C. BrdU incorporation Ј Ј Ј mmup2: 5 -CAGGCTATCCCGCTTAAACACC-3 and ORC6-H-R: 5 - CTAAGCCTC- was detected by using a monoclonal antibody (Becton Dickinson). Immuno- Ј GAGAAGCTGGC-3 . PCR fragments were cloned by using TOPO TA or Zero staining protocols of larval brains and polytene chromosomes have been Blunt TOPO PCR (Invitrogen) cloning kits and sequenced. described previously (18, 36).

Rescue of the Orc6 Mutant. The orc6 native promoter was ampliﬁed by PCR ACKNOWLEDGMENTS. We thank Kirill Popov and Guillermo Marques for from genomic DNA and inserted into the EcoRI site of the pUAS vector helpful discussions and advice. This work is supported by National Institutes of upstream of wild-type or mutant GFP-Orc6 genes. CG1667ϩOrc6 genomic Health Grant GM69681.

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