DNA replication is fundamentally important for tissue development, growth, and homeostasis. Impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Meier–Gorlin Syndrome (MGS) is an autosomal recessive disorder that is also known as ear, patella, short stature syndrome and/or microtia, absent patella, micrognathia syndrome, traits highlighting the core clinical phenotypes (Gorlin et al. 1975; Bicknell et al. 2011a; de Munnik et al. 2012a,b). The genes affected by MGS mutations include many members of prereplicative complex (pre-RC), such as Origin Recognition Complex (ORC) subunits (Orc1, Orc4, Orc6), Cdc6, Cdt1, CDC45, and MCM5, as well as Geminin (Bicknell et al. 2011a,b; Guernsey et al. 2011; Burrage et al. 2015; Fenwick et al. 2016; Vetro et al. 2017; McDaniel et al. 2020), suggesting that the clinical phenotype is caused by defects in DNA replication initiation. As pre-RC complex is essential for DNA replication, the mutations in its components are expected to impair cell proliferation and to reduce growth.

Recently, a new Orc6 mutation was described (Li et al. 2017) that also resulted in MGS. Unlike previously described MGS mutation (Bicknell et al. 2011a), this amino acid substitution (K23E) localizes in the N-terminal domain of Orc6, which is important for DNA binding. In our study, to investigate the functions of different Orc6 domains in a live animal model, we introduced human, Drosophila, and hybrid human-Drosophila orc6 genes into flies with an orc6 null mutant background. Using these “humanized” fly strains carrying the K23E MGS mutation, we identified a molecular mechanism resulting in a MGS phenotype and compared it with our Y225S MGS fly model. We found that despite having different underlying molecular mechanisms, both MGS mutations resulted in similar phenotypes, deficient pre-RC formation, and reduced DNA replication.

Materials and MethodsMutagenesis

Conserved lysine at position 23 was replaced with glutamic acid to create the Meier–Gorlin mutation (K23E), following site-directed mutagenesis protocol (Agilent). Drosophila-human and human-Drosophila hybrids were designed by using the PCR technique. Orc6 mutant and hybrid genes were cloned in frame, with GFP into the modified pUAST vector containing UAS promoter and orc6 Drosophila native promoter. All constructs were injected into Drosophila w1118 embryos (Model System Injections, Raleigh, NC) and individual transgenic strains were maintained.

Purification of recombinant Orc6

Wild-type, mutant, or hybrid Orc6 complementary DNAs (cDNAs) were cloned into the pET15b expression vector, and used to transform Escherichia coli strain BL21. Ni-NTA-purified Orc6 proteins were further purified with HiTrap SP HP (GE Life Sciences) and Superdex 75 (GE Life Sciences) columns.

Electrophoretic mobility shift assay

We incubated 1 ng of 32P end-labeled human B2 lamin or Drosophila ori-β fragments with 300 ng of purified protein in reaction buffer (25 mM Tris, pH 8, 60 mM KCl, 5 mM MgCl2, 0.1% NP-40, 0.12 mg/ml BSA, 10% glycerol) at room temperature for 30 min. Then, 10 µl of each reaction was loaded onto a 4% native polyacrylamide gel. Electrophoresis was performed at room temperature using TAE at pH 8.3 for Drosophila Orc6 and pH 9.5 for human Orc6 as a running buffer. The gel was dried on Whatman paper and exposed to X-ray film.

Fly stocks and rescue experiments

We set up the following fly stocks containing endogenous orc635 deletion and GFP-orc6 fused transposon: (1) orc635/Cy; GFP-Orc6-DmWT: wild type Drosophila Orc6; (2) orc635/Cy; GFP-Orc6-DmK23E: Drosophila Orc6 with K23E MGS mutation; (3) orc635/Cy; GFP-Orc6-HsWT: wild type human Orc6; (4) orc635/Cy; GFP-Orc6-HsK23E: human Orc6 with K23E MGS mutation; (5) orc635/Cy; GFP-Orc6-HD: interspecies hybrid; (6) orc635/Cy; GFP-Orc6-DH: interspecies hybrid; and (7) orc635/Cy; GFP-Orc6-HDK23E: interspecies hybrid with K23E MGS mutation.

Bloomington Drosophila Stock Center stock #5138 (y,w; PLL7/TM3,Sb,Ser) expressing GAL4 ubiquitously under the control of the alphaTub84B promoter was used to design orc635/Cy; tub-GAL4/TM3,Sb fly stocks for rescue experiments.

Fly stock orc635/Cy; GFP-Orc6-DmY225S with Y225S MGS mutation (Balasov et al. 2015) was used as a negative control for immunoprecipitation experiments.

In rescue experiments with a native Drosophila promoter, progeny from heterozygous orc635/Cy; GFP-Orc6 were analyzed for the presence of orc635/orc635; GFP-Orc6 adult flies. In rescue experiments with alphaTub84 (tub) promoter, females of the genotype orc635/Cy; GFP-Orc6 were crossed to males orc635/Cy; tub-GAL4/TM3,Sb, and resulting progeny were analyzed for the presence of orc635/orc635;GFP-Orc6/tub-GAL4 adults.

For Orc6 depletion in motor neurons, we used the Orc6 RNA interference fly stock #HMJ22188 (National Institute of Genetics) (NIG-FLY Stock Center, Japan) and the motor-neuron-specific D42-GAL4 driver #8816 (Bloomington Drosophila Stock Center).

BrdU labeling and immunostaining of third instar larval brains

Larval brains were soaked in PBS with 1 µM BrdU for 30 min at 25°. BrdU incorporation was detected by monoclonal antibodies (Becton Dickinson) following the manufacturer’s protocol, as described previously (Sullivan et al. 2000). Images was made with Olympus Fluoview FV300 confocal microscope. BrdU incorporation was calculated as a percentage of BrdU signal from total brain area using cellSens Dimension Desktop software (Olympus).

Immunostaining of polytene chromosomes

The flies bearing GFP-Orc6 transgene were crossed to Bloomington Drosophila Stock Center fly stock 6870 (w1118; PTP1) expressing GAL4 under Sgs3 promoter. This promoter drives GAL4 in the salivary glands of third instar larvae. Salivary glands expressing different variants of GFP-Orc6 were dissected in PBS with 0.1% NP-40, transferred in fixing solution (2% formaldehyde, 45% acetic acid) for 1 min, squashed in 45% acetic acid, and frozen in liquid nitrogen. The slides were desiccated in 96% alcohol and stored in 70% alcohol at −20°. For immunofluorescence studies, slides were briefly washed in PBS with 0.1% NP-40 and incubated with primary antibodies [mouse anti-GFP (B2), #sc9996; Santa Cruz Biotechnology] diluted in 10% goat antiserum for 2 hr in humid chamber. Secondary goat anti-mouse antibodies (Alexa Fluor 488; Thermo Fisher Scientific) were used to visualize Orc6 on the polytene chromosomes.

Immunoprecipitation from ovaries

Twenty freshly dissected ovaries were crushed in a glass homogenizer with 100 µl of high-salt immunoprecipitation buffer (25 mM HEPES pH 7.6, 12.5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, 300 mM NaCl, 0.01% Triton X-100) and incubated for 1 hr at 4° with continuous rotation. Extract was centrifuged at 15000 × g for 15 min and supernatant was diluted three times with low-salt immunoprecipitation buffer (25 mM HEPES pH 7.6, 12.5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, 0.01% Triton X-100). Protein A-Sepharose (#6501-5; BioVision) and rabbit polyclonal antibodies against Drosophila Orc2 subunit were added and incubated with the supernatant overnight. Sepharose beads were washed three times with low-salt immunoprecipitation buffer and diluted in 10 µl of immunoprecipitation buffer. Samples were boiled in loading buffer, separated in 10% SDS-polyacrylamide gel, and transferred on Immobilon-P membrane (#IPVH00010; Millipore, Bedford, MA). The presence of GFP-Orc6 fused protein was detected with a mouse anti-GFP (B2, #sc9996; Santa Cruz Biotechnology) antibody. A mouse anti-Orc5 antibody was used to verify ORC complex immunoprecipitation.

Mitotic chromosome preparation

Preparation of mitotic nuclei was described previously (Lebedeva et al. 2000). Briefly, third instar larval neural ganglia were incubated in 0.075M KCl for 5 min, fixed in methanol with acetic acid (3:1) for 20 min, and then dispersed in a drop of 50% propionic acid on a slide. Then slides were dried and stained with 5% Giemsa’s solution.

Western blot of larval brains

Freshly dissected brains were incubated with 450 mM NaCl, 0.5% NP-40 in PBS buffer for 1 hr, and samples were centrifuged 10 min at 10000 × g. Pellets were washed three times with 100 mM NaCl, 0.5% NP-40 in PBS buffer and used for Western blotting. MCM complex was detected with rabbit polyclonal anti-MCM4 and anti-MCM5 antibodies.

Fluorescence spectroscopy

Purified Orc6 protein (wild type or mutant) was diluted in buffer (50 mM NaH2PO4, pH 7.4, 100 mM NaCl) to 10 ng/µl. Then, 260 µl samples were loaded in quartz cuvette. Fluorescence measurements were performed in the VARIAN CARY Eclipse fluorescence spectrophotometer. The spectral widths of the excitation and the emission bands were 5 and 20 nm, respectively. Excitation was performed at 290 nm wavelength. Emission spectra were recorded from 300 to 400 nm in 1-nm steps. An emission spectrum of a buffer was subtracted from protein spectra.

Microarray analysis

RNA was isolated from 5-day-old females using ZR Tissue and Insect RNA MicroPrep (#R2030; Zymo Research). DNA was removed using TURBO DNase (#AM2238; Invitrogen, Carlsbad, CA) following manufacturer’s recommendations. cDNA was generated from 1 μg of total RNA using ProtoScript II First Strand cDNA Synthesis Kit (#E6560; New England Biolabs).

The microarray experiment was conducted at the Boston University Microarray and Sequencing Resource Core Facility. Drosophila Gene 1.0 ST CEL files were normalized to produce gene-level expression values using the implementation of the robust multiarray average (Irizarry et al. 2003) in the affy package (version 1.48.0) (Gautier et al. 2004) included in the Bioconductor software suite (version 3.2) (Gentleman et al. 2004), and an Entrez Gene-specific probeset mapping (20.0.0) from the Molecular and Behavioral Neuroscience Institute (Brainarray) at the University of Michigan (Dai et al. 2005) (brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF). Array quality was assessed by computing relative log expression and normalized unscaled standard error, using the affyPLM package (version 1.46.0). Principal component analysis was performed using the prcomp R function with expression values that had been normalized across all samples to a mean of zero and a standard deviation of one. Differential expression was assessed using the moderated (empirical Bayesian) t-test implemented in the limma package (version 3.26.9) (i.e., creating simple linear models with lmFit, followed by empirical Bayesian adjustment with eBayes). Correction for multiple hypothesis testing was accomplished using the Benjamini–Hochberg false discovery rate. Human homologs of fly genes were identified using HomoloGene (version 68). All microarray analyses were performed using the R environment for statistical computing (version 3.2.0). Gene ontology terms analysis was conducted using DAVID Bioinformatics Resources 6.8 (david.ncifcrf.gov) (Huang et al. 2009).

Data availability statement

Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Supplemental material available at figshare: https://doi.org/10.25386/genetics.13070288.

ResultsEstablishing Drosophila strains to study the functions of human Orc6 in vivo

The metazoan Orc6 consists of two domains (Figure 1): a larger N-terminal domain that carries a homology with TFIIB transcription factor and is important for DNA binding (Chesnokov et al. 2003; Balasov et al. 2007; Liu et al. 2011), and a shorter C-terminal domain essential for the interaction of the protein with the core ORC through its Orc3 subunit (Bleichert et al. 2013). The C-terminal domain of metazoan Orc6 is also important for the function of Orc6 in cytokinesis (Prasanth et al. 2002; Chesnokov et al. 2003; Huijbregts et al. 2009; Bernal and Venkitaraman 2011; Akhmetova et al. 2015).

Figure 1

Domain organization of metazoan Orc6 protein. Orc6 protein consists of the N-terminal TFIIB-like domain important for DNA binding, and C-terminal domain required for the interaction with core ORC. Drosophila and human N-terminal domains share 27% amino acid identity and 48% similarity. C-terminal domains have only 15% amino acid identity and 22% similarity. The resulting amino acid changes from the new MGS mutation, the conservative lysine at position 23 (K23E), as well as the previously described C-terminal MGS mutation (Y225S) are indicated in red.

In our previous work we generated a deletion of the orc6 gene in Drosophila allowing the studies of the protein in vivo. We showed that the orc6 deletion resulted in defects in DNA replication as well as abnormal chromosome condensation and segregation (Balasov et al. 2009). Orc6 is the least conserved among ORC subunits, with metazoan Orc6 proteins showing significantly higher homology as compared to budding yeast protein. The full-length human orc6 gene (Orc6-HsWT) under control of native orc6 promoter did not rescue lethality of the Orc6 deficient flies (Balasov et al. 2009). However, human Orc6 protein was able to bind chromosomes when expressed in Drosophila (Liu et al. 2011). We asked if the expression of a hybrid protein designed from the human DNA binding domain of Orc6 and the Drosophila C terminus responsible for the interaction with a core ORC might rescue the lethality associated with the Orc6 deletion. Two constructs were created (Figure 2A). The first consisted of the Drosophila N-terminal TFIIB-like domain and human C terminus (Orc6-DH); the second construct contained human N-terminal domain and Drosophila C terminus (Orc6-HD). Both constructs were tested for their ability to rescue orc6 deletion. The expression of Orc6-DH did not rescue the lethality; however, the expression of Orc6-HD resulted in a full rescue and obtained flies were undistinguishable from the flies rescued with wild-type Drosophila Orc6 (Orc6-DmWT) (Figure 2A).

Figure 2

Design of hybrid Orc6 and Orc6 mutants. (A) Diagrams of transgenic constructs. Black represents human Orc6 and red represents Drosophila Orc6. Orc6-DmWT and Orc6-HsWT indicate wild-type Drosophila and human proteins, respectively. Orc6-DH is the hybrid protein containing first 205 amino acids of Drosophila and last 46 amino acids of human Orc6. Orc6-HD is the hybrid protein containing first 206 amino acids of human and last 52 amino acids of Drosophila Orc6. (B) Immunoprecipitation of the ORC complex with anti-Orc2 antibodies from ovaries expressing GFP-tagged Orc6 shown in A. (C) The position of the K23E substitution in Drosophila Orc6 (red). (D) Immunoprecipitation of ORC complex using Orc2 antibodies from Drosophila Orc6-K23E mutant fly ovaries. Immunoprecipitations from three independent transgenic fly stocks are shown: DmK23E-3, DmK23E-4, and DmK23E-6. (E) The position of the K23E in hybrid human-Drosophila Orc6. The substitution is colored in red. (F) Immunoprecipitation of ORC complex using Orc2 antibodies from hybrid human-Drosophila K23E mutant fly ovaries. Immunoprecipitations from three independent transgenic fly stocks are shown: HDK23E-4, HDK23E-6, and HDK23E-9. Orc6-DmY225S was used as a negative control.

Orc6 is a subunit of the ORC complex. Therefore, we analyzed the ability of Orc6 proteins described above to associate with Drosophila ORC complex. We employed anti-Orc2 antibodies for immunoprecipitation of ORC complex, and antibodies against Orc5 subunit were used as a rigorous control for the presence of the whole ORC complex in immunoprecipitated material. We found that Orc6-HD but not Orc6-DH or wild-type human Orc6-HsWT immunoprecipitated with the rest of the ORC complex (Figure 2B).

Furthermore, the MCM helicase association with the chromatin was diminished in Orc6-DH as compared to the Orc6-HD strain (Supplemental Material, Figure S1A), indicating that the pre-RC formation is impaired in the presence of Orc6-DH form of the protein.

Since Orc6-HD compensated for the orc6 deletion similar to the wild-type Drosophila Orc6, this transgene can be used for studying mutations in the human large N-terminal TFIIB-like domain portion of Orc6 protein in the live animal model.

The analysis of flies carrying MGS mutation localized in the N-terminal domain of Orc6

In our previous analysis of Orc6 functions in vivo, we showed that the mutation of the conserved tyrosine to serine (Y225S) in the C-terminal domain of Drosophila Orc6 resulted in third instar lethality associated with defects in DNA replication and chromosome abnormalities (Balasov et al. 2015). In humans, the corresponding mutation (Y232S) is associated with MGS. The molecular analyses of the mutation in this Drosophila model of MGS revealed that it disrupted the interaction between Orc6 and the rest of the ORC through the Orc3 subunit, thereby impairing pre-RC formation and MCM recruitment, which is essential for DNA replication (Bleichert et al. 2013; Balasov et al. 2015).

Recently, the first diagnosed MGS case in China was described (Li et al. 2017). The patient had a short stature, microtia, small patella, and craniofacial abnormalities characteristic for MGS. It was reported that the patient carried a novel homozygous mutation in the orc6 gene, resulting in a change of A to G at position 67, which corresponds to the conversion of lysine at position 23 to glutamic acid (K23E). Interestingly, this mutation is localized within N-terminal TFIIB-like domain of the protein, unlike the earlier described Y232S MGS mutation (Bicknell et al. 2011a; de Munnik et al. 2012a), which effects the C-terminal domain of the protein (Figure 1).

We then set about determining the molecular consequences of the new MGS mutation. Since only wild-type Drosophila Orc6 and hybrid Orc6-HD resulted in viable adults, we incorporated K23E mutation into Drosophila Orc6 gene and human N-terminal domain of the Orc6-HD hybrid construct (Figure 2, C and E). The resulting constructs, Orc6–DmK23E and Orc6-HDK23E, were introduced into flies carrying the homozygous deletion of the orc6 gene. Three independent transgenic fly stocks were set up for each mutant construct (Figure S2).

To analyze the ability of mutant proteins to interact with the rest of the complex, we immunoprecipitated ORC from obtained transgenic flies using Orc2 antibodies. Drosophila Orc6 carrying Y225S mutation (Orc6-DmY225S) was used as a negative control for complex association since this mutation abolished the interaction of Orc6 protein with ORC complex (Balasov et al. 2015). As expected, Orc6-DmY225S protein did not immunoprecipitate with ORC (Figure 2, D and F). However, both Drosophila and hybrid Human-Drosophila Orc6 proteins carrying the K23E substitution were present in the immunoprecipitated material indicating that the interaction with core ORC was not affected by the mutation (Figure 2, D and F). It was previously shown that metazoan Orc6 associated with ORC via its Orc3 subunit (Bleichert et al. 2013). Therefore, we tested whether K23E mutation would disrupt a direct interaction between Orc6 and Orc3 subunits. As shown in Figure S3, mutant Orc6 protein carrying the K23E mutation was able to pull down Orc3 in the in vitro transcription/translation reaction as well as the wild-type Drosophila Orc6. We conclude that K23E mutation in Orc6 does not affect its association with the core ORC.

Replication defects often result in a loss of chromosome integrity. Therefore, we investigated mitotic chromosomes in developing larval brains, since this tissue is known to provide the best mitotic figures. The normal Drosophila melanogaster karyotype consists of four pairs of chromosomes (Figure 3A, Orc6-DmWT). In larvae carrying orc6 deletion (Figure 3A, Orc6 deletion) or expressing Orc6-DH (Figure 3A, Orc6-DH) or Orc6-HsWT (Figure 3C, Orc6-HsWT) constructs on orc6 deletion background, chromosomes lost parts of their arms, appeared aberrantly condensed, and fragmented. On the other hand, Orc6-HD transgene expression restored mitotic chromosome structure (Figure 3A, Orc6-HD). The incorporation of the K23E mutation into Orc6 resulted in chromosome defects and fragmentations (Figure 3, B and C). To quantify chromosome integrity, we counted percentages of severely affected mitoses (as in Orc6 deletion mutant), mildly/moderately affected (as in Orc6-HDK23E-4), and normal mitoses (as in Orc6-DmWT) (Figure 3D). As expected, two transgenic stocks, Orc6-DmWT and Orc6-HD, restored all karyotypes (100%) to the normal pattern. Orc6 deletion, Orc6-DH, and Orc6-HsWT contained >95% of defective mitoses. Orc6-DmK23E and Orc6-HDK23E mutants all contained some defective mitoses with different degree of severity (Figure 3D). The minor chromosome defects were also reported for the human patient carrying the K23E mutation (Li et al. 2017).

Figure 3

Mitoses in larval neuroblasts. Brains of third instar larvae expressing specified transgenes on orc6 deletion background were analyzed for the karyotype. (A) Orc6 deletion mutant and hybrid Orc6 (Orc6-DH, Orc6-HD) compared to Drosophila wild type (Orc6-DmWT). (B) Three independent DmOrc6 transgenics bearing the K23E mutation (Orc6-DmK23E-3, Orc6-DmK23E-4, Orc6-DmK23E-6). (C) Three independent hybrid Orc6-HD transgenic lines bearing the mutation in human portion of Orc6 (Orc6-HDK23E-4, Orc6-HDK23E-6, Orc6-HDK23E-9) compared to human wild-type Orc6 (Orc6-HsWT). Bars represents 1 µm. (D) Percentages of defective (orange and red) and normal (green) karyotypes. Total number of mitoses analyzed is shown above the chart.

To investigate replication defects more rigorously, we analyzed BrdU incorporation in larval brains of the fly stocks expressing hybrid or mutant proteins. Brains were underdeveloped and BrdU incorporation was reduced in flies carrying the Orc6-HsWT or Orc6-DH constructs (Figure 4, B and4D). No difference was observed between the Orc6-DmWT and Orc6-HD hybrid constructs (Figure 4, A and C). The larval brains of the fly stocks expressing mutant proteins Orc6-DmK23E (Figure 4E) or Orc6-HDK23E (Figure 4F) were also underdeveloped and showed reduced BrdU labeling. Quantifications confirmed that Orc6-HD was not statistically different from Orc6-DmWT, whereas all other analyzed mutants were defective (Figure 4G).

Figure 4

BrdU incorporation in larval brains. Brains of third instar larvae expressing specified transgenes on orc6 deletion background were labeled with BrdU. Transgenes are as follows: (A) wild-type Drosophila Orc6, (B) Orc6-DH hybrid, (C) Orc6-HD hybrid, (D) wild-type human Orc6, (E) Drosophila Orc6-K23E mutant, and (F) Orc6-HD hybrid with the K23E mutation. Bars represents 50 µm. (G) Percentages of BrdU-labeled area relative to total brain area (n = 5). * indicates a statistically significant difference compared to Orc6-DmWT; ns indicates no significant difference.

Next, we calculated the percentage of homozygous flies rescued with different transgenes driven by native Orc6 promoter (Table 1). We found that stock Orc6-DmK23E-4 resulted in 28% of adult viability. The majority of rescued flies (82%) were indistinguishable from wild type (Figure 5A). Minor defects (missing scutellar bristles) appeared in 18% of flies (Figure 5B). It should be noted that stock Orc6-DmK23E-4 had the highest expression level of Orc6-DmK23E protein compared to others, and promoted flies to the adult stage (Figure S2 and Table 1). Another stock Orc6-DmK23E-6 produced very few (3%) homozygous adults (Table 1). All of the surviving flies had defective scutellar bristles and reduced eye size (Figure 5C). The eye of Drosophila is a well-established model to study proliferation defects. The compound eye of Drosophila consists of uniform regular facets, and the final number of them is determined during several mitotic divisions in third instar larvae. Any delay or disturbance during the course of divisions result in reduced size and abnormal shape of the adult eye. This phenotype was consistent with a disrupted Orc6 function in replication and cell cycle progression. Another interesting observed phenotype was the inability of mutant flies to fly. Rescued adult flies carrying Orc6-DmK23E could not fly but were able to jump and walk. Previously we reported that flies with C-terminal Orc6 MGS mutation (Y225S) also missed scutellar bristles and were flightless (Balasov et al. 2015). The observed bristle defect and flightless phenotype could be a common MGS feature in Drosophila.

Table 1 Rescue of the orc6 deletion with the expression of different Orc6 transgenes under control of native or tubulin promoterFigure 5

Phenotypes of orc6 deletion flies rescued with Orc6-DmK23E. (A) Flies expressing wild-type Drosophila Orc6. (B) Orc6DmK23E-4 mutant flies have mild phenotype: eye size and facets number are normal; however, 18% of flies have missing or defective scutellar bristles (arrows). (C) All surviving flies carrying Orc6DmK23E-6 showed reduced eye facets number and multiple defects of scutellar bristles (arrows).

In our previous study describing our fly model of MGS based on Orc6 Y225S mutation (Balasov et al. 2015), we showed that the elevated expression of Orc6 Y225S transgene rescued the orc6 deletion phenotype. We investigated whether it would be the case for K23E mutation. Similar to the Y225S Orc6 mutant, all K23E orc6 transgenes have an UAS promoter upstream of native orc6 promoter. The UAS promoter allows increased Orc6 transgene expression by crossing to flies bearing the tubulin promoter-driven GAL4 (see Materials and Methods). As summarized in Table 1, elevated expression of both Drosophila and human-Drosophila hybrid transgenes carrying the K23E mutation promoted flies to adult stage. Remarkably, these rescued flies also had defects of the scutellar bristles and were not able to fly, similar to the phenotypes observed in the fly model of MGS based on the Orc6 Y225S mutation (Balasov et al. 2015).

Overall, we concluded that both MGS mutations resulted in reduced DNA replication and chromosome defects, manifesting in similar phenotypes; however, the molecular mechanisms driving this phenotype might be different. The Y225S substitution impedes Orc6 binding to the ORC complex (Bleichert et al. 2013; Balasov et al. 2015). The K23E substitution resides in the N-terminal domain of Orc6 that is important for DNA binding. Therefore, we investigated the ability of Drosophila, human, and hybrid Orc6 proteins carrying the K23E mutation to bind DNA.

K23E mutations disrupt DNA binding ability of Orc6 protein

Six recombinant proteins were purified using an E. coli expression system: Drosophila (Orc6-DmWT and Orc6-DmK23E), hybrid human/Drosophila (Orc6-HD and Orc6-HDK23E), and human (Orc6-HsWT and Orc6-HsK23E) (Figure S4A). As shown in Figure 6A, both Drosophila Orc6 wild-type protein and Orc6 carrying the K23E mutation were able to bind in vitro with the ori-β DNA fragment derived from the origin of DNA replication associated with a chorion gene cluster. Binding was very tight, with the majority of the labeled probe bound by the protein, as we observed before (Balasov et al. 2007), suggesting the presence of multiple binding sites for Orc6 within the ori-β fragments. However, the mobility of DNA-protein complex changed significantly when Orc6-K23E was used in the reaction. The mutant complex migrated faster compared to the wild-type protein, probably due to a reduced template occupancy resulting from the K23E mutation. Increasing the concentration of the mutant protein in electrophoretic mobility shift assay (EMSA) reactions slightly improved the DNA binding of Orc6-K23E protein (Figure S4B).

Figure 6

DNA and chromosome binding ability of hybrid and mutant Orc6. Electrophoretic mobility shift assay (EMSA) and chromosome binding of (A and D) Drosophila Orc6 wild type and K23E mutant; (B and E) hybrid Orc6 HD wild type and Orc6 HD-K23E mutant proteins; and (C and F) human Orc6 wild type and K23E mutant. “50°C 15 min” indicates that protein probe was heat-treated before adding to the reaction. The amount of competitor poly(dI-dC) and poly(dG-dC) is shown in nanograms. Polytene chromosomes isolated from salivary glands expressing GFP-tagged Orc6 were immunostained with anti-GFP antibodies, and DNA was stained with DAPI.

When the K23E substitution was made in the human Orc6 protein or in a hybrid protein Orc6-HD containing human N-terminal TFIIB-like domain, the overall DNA binding ability of the resulting mutant proteins was significantly diminished (Figure 6, B and C). We would like to point out that the DNA binding pattern for human Orc6 is different from Drosophila protein, with the majority of the DNA-protein complex migrating at the top of the EMSA gel. This difference between fly and human Orc6 binding to DNA was reported previously (Balasov et al. 2007; Liu et al. 2011).

To visualize the Orc6 mutants binding in vivo, we used GFP-Orc6 bearing fly stocks described in Materials and Methods. The UAS promoter in the construct allows for GAL4-induced expression using the GAL4/UAS binary system (Brand and Perrimon 1993; Duffy 2002). GFP-Orc6 expression was induced in salivary glands of third instar larvae to test chromosome binding of Orc6 mutants. Nuclei of Drosophila salivary glands contain polytene chromosomes that can be easily visualized with microscopy. Both Gal4-induced Orc6-DmWT and Orc6-DmK23E (Figure 6D) were found to be associated with polytene chromosomes. In contrast, over-expressed Orc6-HDK23E and Orc6-HsK23E mutants showed significantly reduced DNA binding and association with chromosomes (Figure 6, E and F), in agreement with the EMSA DNA binding experiments shown in Figure 6, B and C. Together, our data on DNA binding suggest that K23E mutation has a debilitating effect on DNA binding ability of Orc6.

K23E mutation results in the instability of the Drosophila, but not human, Orc6

Often mutations lead to a protein unfolding that may affect its functions, such as DNA binding. Therefore, we tested the effect of the K23E substitution on protein stability. In this experiment the unfolding of the protein was studied by measuring the intrinsic fluorescence intensity at a wavelength of excitation corresponding to tryptophan. Tryptophan fluorescence is very sensitive to protein conformational changes and thus provides information about changes in secondary and/or tertiary structure. A decrease in fluorescence intensity and shift in the maximal emission wavelength reflect protein unfolding (Royer 2006). We measured the tryptophan fluorescence of Drosophila and human Orc6 proteins at the increasing temperatures and compared them with mutants carrying the K23E mutation. As presented in Figure 7A, the K23E mutation in human Orc6 did not change a stability of the mutant protein relative to wild type at all tested temperatures; however, the Drosophila Orc6 carrying the K23E mutation was more susceptible to the heat and began unfolding at temperatures over 35°, suggesting that the amino acid substitution causes an instability in fly protein. Importantly, fluorescence intensity of the Drosophila mutant relative to wild type was significantly lower even at 25° (Figure 7B). This indicates that some part of the mutant protein is already unfolded at temperatures optimal for Drosophila development. We hypothesized that elevated temperature may increase protein unfolding and further affect the DNA binding ability of Drosophila Orc6. To test this hypothesis we repeated EMSA with wild-type and K23E mutant protein probes heat shocked at 50° for 15 min. Mutant Orc6 did not show any DNA binding under these conditions, whereas the wild-type Orc6 bound DNA but migrated faster compared to the protein not subjected to heat treatment (Figure 6A and Figure S4B). We suggest that the instability of Orc6-DmK23E protein contributes to the loss of DNA binding ability and ultimately to the MGS phenotype in Drosophila.

Figure 7

Tryptophan fluorescence of Drosophila and human Orc6 proteins. (A) Emission maximum wavelengths at gradually increasing temperatures are shown. Shift in the maximal emission wavelength reflects protein unfolding. (B) Absolute intensity at each temperature point. DmWT and DmK23E indicate Drosophila Orc6 wild type and Orc6-K23E mutant, respectively, and HsWT and HsK23E indicate human Orc6 wild type and Orc6 HsK23E mutant, respectively.

Elevated expression of the wild-type human Orc6 in Drosophila rescues a lethality associated with deletion of the orc6 gene

In our previous work (Balasov et al. 2015), we found that Drosophila C-terminal MGS mutation Y225S was lethal in flies despite the mild MGS clinical appearance in humans. Importantly, human Orc6 is loosely associated with the core ORC (Vashee et al. 2001; Vashee et al. 2003; Ranjan and Gossen 2006), whereas Drosophila Orc6 associates with core ORC much more tightly (Gossen et al. 1995; Chesnokov et al. 1999; Chesnokov et al. 2001). This might explain why the Y225S MGS mutation in Orc6 has a more dramatic effect on survival in Drosophila compared to the corresponding Y232S mutation in humans. We also found that elevated expression of mutant protein carrying Y225S substitution rescued lethality, restored normal karyotype, and allowed detection of Orc6 with the rest of ORC complex (Balasov et al. 2015). The expression of the wild-type human Orc6 by the native orc6 promoter could not rescue a lethality associated with Drosophila orc6 gene deletion (Table 1). We asked if overexpression of the human Orc6 would rescue orc6 deficiency in Drosophila. Using a GAL4/UAS binary system, we boosted the expression of human wild type and K23E mutant in flies. We found that the elevated expression of the wild-type human Orc6 was able to rescue flies to viability (Table 1) with restoration of normal karyotype (Figure 8A). We also observed human Orc6 with other ORC subunits after immunoprecipitation with anti-Orc2 antibodies (Figure 8B). Remarkably, rescued adult flies displayed an upheld wing phenotype (Figure 8, C and D) and were not able to fly (File S1, movie file), similar to the Drosophila C-terminal MGS mutation Y225S flies (Balasov et al. 2015). However, when K23E mutation was introduced into the human Orc6, the resulting mutant Orc6-HsK23E transgene was not able to rescue orc6 deficient flies to viability even under overexpressed conditions (Table 1), and no improvements were observed during development of the flies (Figure 8A, lower row). The loose association of human Orc6 with the core complex somewhat mimics the disruptive effect of Y225S substitution in Drosophila Orc6 for interaction with ORC. As a result, the Orc6-HsK23E mutant appears to be defective in both DNA binding (Figure 6, C and F) and in interacting with the core ORC, therefore it was not possible to rescue a lethality regardless of the protein expression level (Table 1).