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 meta- phase, 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.