HIGHLIGHTED ARTICLE

| INVESTIGATION

Humanized Drosophila Model of the Meier-Gorlin

Syndrome Reveals Conserved and Divergent Features
of the Orc6 Protein
Maxim Balasov, Katarina Akhmetova, and Igor Chesnokov1
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, Alabama 35294
ORCID ID: 0000-0002-6659-2913 (I.C.)

ABSTRACT Meier–Gorlin syndrome (MGS) is a rare, autosomal recessive disorder characterized by microtia, primordial dwarfism, small
ears, and skeletal abnormalities. Patients with MGS often carry mutations in genes encoding the subunits of the Origin Recognition
Complex (ORC), components of the prereplicative complex and replication machinery. Orc6 is an important component of ORC and
has functions in both DNA replication and cytokinesis. A mutation in the conserved C-terminal motif of Orc6 associated with MGS
impedes the interaction of Orc6 with core ORC. Recently, a new mutation in Orc6 was also identified; however, it is localized in the
N-terminal domain of the protein. To study the functions of Orc6, we used the human gene to rescue the orc6 deletion in Drosophila.
Using this “humanized” Orc6-based Drosophila model of MGS, we discovered that unlike the previous Y225S MGS mutation in Orc6,
the K23E substitution in the N-terminal TFIIB-like domain of Orc6 disrupts the protein ability to bind DNA. Our studies revealed the
importance of evolutionarily conserved and variable domains of Orc6 protein, and allowed the studies of human protein functions and
the analysis of the critical amino acids in live animal heterologous system, as well as provided novel insights into the mechanisms
underlying MGS pathology.
KEYWORDS DNA replication; Drosophila; Meier–Gorlin syndrome; ORC

D NA replication is fundamentally important for tissue

development, growth, and homeostasis. Impairments
of the DNA replication machinery can have catastrophic
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
consequences for genome stability and cell division. Meier– DNA replication, the mutations in its components are ex-
Gorlin Syndrome (MGS) is an autosomal recessive disorder pected to impair cell proliferation and to reduce growth.
that is also known as ear, patella, short stature syndrome ORC plays a central role in the initiation of DNA replication
and/or microtia, absent patella, micrognathia syndrome, but is also involved in nonreplicative functions (Bell and
traits highlighting the core clinical phenotypes (Gorlin Stillman 1992; Bell 2002; Chesnokov 2007; Sasaki and
et al. 1975; Bicknell et al. 2011a; de Munnik et al. 2012a,b). Gilbert 2007). The smallest subunit of ORC, Orc6 is the most
The genes affected by MGS mutations include many mem- divergent and enigmatic among ORC subunits. Orc6 is im-
bers of prereplicative complex (pre-RC), such as Origin Rec- portant for DNA replication in all species (Lee and Bell 1997;
ognition Complex (ORC) subunits (Orc1, Orc4, Orc6), Cdc6, Chesnokov et al. 2001; Chesnokov et al. 2003; Semple et al.
Cdt1, CDC45, and MCM5, as well as Geminin (Bicknell 2006; Balasov et al. 2007; Chen et al. 2007; Balasov et al.
et al. 2011a,b; Guernsey et al. 2011; Burrage et al. 2015; 2009). It is also essential for cytokinesis in Drosophila and
human cells (Prasanth et al. 2002; Chesnokov et al. 2003;
Copyright © 2020 by the Genetics Society of America
doi: https://doi.org/10.1534/genetics.120.303698 Bernal and Venkitaraman 2011). In Drosophila, Orc6 stimu-
Manuscript received June 5, 2020; accepted for publication October 6, 2020; published lates septin complex GTPase activity and polymerization dur-
Early Online October 9, 2020.
Supplemental material available at figshare: https://doi.org/10.25386/genetics.
ing filament assembly through protein-protein interactions
13070288. (Huijbregts et al. 2009; Akhmetova et al. 2015). Metazoan
1
Corresponding author: Department of Biochemistry and Molecular Genetics, School of
Medicine, University of Alabama at Birmingham, 720 20th St. South, Birmingham, AL
Orc6 proteins consist of two functional domains: a larger
35294. E-mail: [email protected] N-terminal domain important for binding of DNA and smaller

Genetics, Vol. 216, 995–1007 December 2020 995

C-terminal domain important for protein-protein interactions Electrophoretic mobility shift assay
(Chesnokov et al. 2003; Balasov et al. 2007; Duncker et al.
We incubated 1 ng of 32P end-labeled human B2 lamin or
2009; Bleichert et al. 2013). It has been shown that in meta-
Drosophila ori-b fragments with 300 ng of purified protein in
zoan species, N-terminal domain of Orc6 shares structural
reaction buffer (25 mM Tris, pH 8, 60 mM KCl, 5 mM MgCl2,
homology with the transcription factor TFIIB (Chesnokov
0.1% NP-40, 0.12 mg/ml BSA, 10% glycerol) at room tem-
et al. 2003; Balasov et al. 2007; Liu et al. 2011). The con-
perature for 30 min. Then, 10 ml of each reaction was loaded
served motif in the C terminus of Orc6 is responsible for
onto a 4% native polyacrylamide gel. Electrophoresis was
interacting with the Orc3 subunit of ORC (Bleichert et al.
performed at room temperature using TAE at pH 8.3 for
2013). A mutation coding for a tyrosine 232 to serine alter-
Drosophila Orc6 and pH 9.5 for human Orc6 as a running
ation in this region of the protein is linked to the MGS in
buffer. The gel was dried on Whatman paper and exposed
humans (Bicknell et al. 2011a; de Munnik et al. 2012a). In
to X-ray film.
our earlier studies, we introduced MGS mutation in Drosophila
Orc6 (Y225S) and established a fruit fly model of MGS. Fly stocks and rescue experiments
These mutant flies displayed severe replication and de- We set up the following fly stocks containing endogenous
velopmental defects due to impaired interaction of mu- orc635 deletion and GFP-orc6 fused transposon: (1) orc635/Cy;
tant Orc6 protein with the rest of the ORC (Balasov et al. GFP-Orc6-DmWT: wild type Drosophila Orc6; (2) orc635/Cy;
2015). GFP-Orc6-DmK23E: Drosophila Orc6 with K23E MGS muta-
Recently, a new Orc6 mutation was described (Li et al. tion; (3) orc635/Cy; GFP-Orc6-HsWT: wild type human Orc6;
2017) that also resulted in MGS. Unlike previously described (4) orc635/Cy; GFP-Orc6-HsK23E: human Orc6 with K23E
MGS mutation (Bicknell et al. 2011a), this amino acid sub- MGS mutation; (5) orc635/Cy; GFP-Orc6-HD: interspecies hy-
stitution (K23E) localizes in the N-terminal domain of Orc6, brid; (6) orc635/Cy; GFP-Orc6-DH: interspecies hybrid; and (7)
which is important for DNA binding. In our study, to investi- orc635/Cy; GFP-Orc6-HDK23E: interspecies hybrid with K23E
gate the functions of different Orc6 domains in a live animal MGS mutation.
model, we introduced human, Drosophila, and hybrid human- Bloomington Drosophila Stock Center stock #5138 (y,w;
Drosophila orc6 genes into flies with an orc6 null mutant P{w+mC = tubP-GAL4}LL7/TM3,Sb,Ser) expressing GAL4
background. Using these “humanized” fly strains carrying ubiquitously under the control of the alphaTub84B promoter
the K23E MGS mutation, we identified a molecular mecha- was used to design orc635/Cy; tub-GAL4/TM3,Sb fly stocks for
nism resulting in a MGS phenotype and compared it with our rescue experiments.
Y225S MGS fly model. We found that despite having differ- Fly stock orc635/Cy; GFP-Orc6-DmY225S with Y225S MGS
ent underlying molecular mechanisms, both MGS mutations mutation (Balasov et al. 2015) was used as a negative control
resulted in similar phenotypes, deficient pre-RC formation, for immunoprecipitation experiments.
and reduced DNA replication. In rescue experiments with a native Drosophila promoter,
progeny from heterozygous orc635/Cy; GFP-Orc6 were ana-
Materials and Methods lyzed for the presence of orc635/orc635; GFP-Orc6 adult flies.
In rescue experiments with alphaTub84 (tub) promoter, fe-
Mutagenesis
males of the genotype orc635/Cy; GFP-Orc6 were crossed to
Conserved lysine at position 23 was replaced with gluta- males orc635/Cy; tub-GAL4/TM3,Sb, and resulting progeny
mic acid to create the Meier–Gorlin mutation (K23E), were analyzed for the presence of orc635/orc635;GFP-Orc6/
following site-directed mutagenesis protocol (Agilent). tub-GAL4 adults.
Drosophila-human and human-Drosophila hybrids were For Orc6 depletion in motor neurons, we used the Orc6
designed by using the PCR technique. Orc6 mutant and RNA interference fly stock #HMJ22188 (National Institute
hybrid genes were cloned in frame, with GFP into the of Genetics) (NIG-FLY Stock Center, Japan) and the motor-
modified pUAST vector containing UAS promoter and neuron-specific D42-GAL4 driver #8816 (Bloomington Dro-
orc6 Drosophila native promoter. All constructs were in- sophila Stock Center).
jected into Drosophila w1118 embryos (Model System In-
BrdU labeling and immunostaining of third instar
jections, Raleigh, NC) and individual transgenic strains
larval brains
were maintained.
Larval brains were soaked in PBS with 1 mM BrdU for 30 min
Purification of recombinant Orc6
at 25°. BrdU incorporation was detected by monoclonal an-
Wild-type, mutant, or hybrid Orc6 complementary DNAs tibodies (Becton Dickinson) following the manufacturer’s
(cDNAs) were cloned into the pET15b expression vector, protocol, as described previously (Sullivan et al. 2000). Im-
and used to transform Escherichia coli strain BL21. Ni-NTA- ages was made with Olympus Fluoview FV300 confocal mi-
purified Orc6 proteins were further purified with HiTrap SP croscope. BrdU incorporation was calculated as a percentage
HP (GE Life Sciences) and Superdex 75 (GE Life Sciences) of BrdU signal from total brain area using cellSens Dimension
columns. Desktop software (Olympus).

996 M. Balasov, K. Akhmetova, and I. Chesnokov

Immunostaining of polytene chromosomes Western blotting. MCM complex was detected with rabbit
polyclonal anti-MCM4 and anti-MCM5 antibodies.
The flies bearing GFP-Orc6 transgene were crossed to Bloo-
mington Drosophila Stock Center fly stock 6870 (w1118; Fluorescence spectroscopy
P{Sgs3-GAL4.PD}TP1) expressing GAL4 under Sgs3 promoter.
Purified Orc6 protein (wild type or mutant) was diluted in
This promoter drives GAL4 in the salivary glands of third
buffer (50 mM NaH2PO4, pH 7.4, 100 mM NaCl) to 10 ng/ml.
instar larvae. Salivary glands expressing different variants
Then, 260 ml samples were loaded in quartz cuvette. Fluo-
of GFP-Orc6 were dissected in PBS with 0.1% NP-40, trans-
rescence measurements were performed in the VARIAN
ferred in fixing solution (2% formaldehyde, 45% acetic acid)
CARY Eclipse fluorescence spectrophotometer. The spectral
for 1 min, squashed in 45% acetic acid, and frozen in liquid
widths of the excitation and the emission bands were 5 and
nitrogen. The slides were desiccated in 96% alcohol and
20 nm, respectively. Excitation was performed at 290 nm
stored in 70% alcohol at 220°. For immunofluorescence
wavelength. Emission spectra were recorded from 300 to
studies, slides were briefly washed in PBS with 0.1% NP-40
400 nm in 1-nm steps. An emission spectrum of a buffer
and incubated with primary antibodies [mouse anti-GFP
was subtracted from protein spectra.
(B2), #sc9996; Santa Cruz Biotechnology] diluted in 10%
goat antiserum for 2 hr in humid chamber. Secondary goat Microarray analysis
anti-mouse antibodies (Alexa Fluor 488; Thermo Fisher RNA was isolated from 5-day-old females using ZR Tissue
Scientific) were used to visualize Orc6 on the polytene and Insect RNA MicroPrep (#R2030; Zymo Research). DNA
chromosomes. was removed using TURBO DNase (#AM2238; Invitrogen,
Immunoprecipitation from ovaries Carlsbad, CA) following manufacturer’s recommendations.
cDNA was generated from 1 mg of total RNA using Proto-
Twenty freshly dissected ovaries were crushed in a glass Script II First Strand cDNA Synthesis Kit (#E6560; New Eng-
homogenizer with 100 ml of high-salt immunoprecipitation land Biolabs).
buffer (25 mM HEPES pH 7.6, 12.5 mM MgCl2, 100 mM KCl, The microarray experiment was conducted at the Boston
0.1 mM EDTA, 300 mM NaCl, 0.01% Triton X-100) and in- University Microarray and Sequencing Resource Core Facility.
cubated for 1 hr at 4° with continuous rotation. Extract was Drosophila Gene 1.0 ST CEL files were normalized to produce
centrifuged at 15000 3 g for 15 min and supernatant was gene-level expression values using the implementation of the
diluted three times with low-salt immunoprecipitation buffer robust multiarray average (Irizarry et al. 2003) in the affy
(25 mM HEPES pH 7.6, 12.5 mM MgCl2, 100 mM KCl, package (version 1.48.0) (Gautier et al. 2004) included in
0.1 mM EDTA, 0.01% Triton X-100). Protein A-Sepharose the Bioconductor software suite (version 3.2) (Gentleman
(#6501-5; BioVision) and rabbit polyclonal antibodies against et al. 2004), and an Entrez Gene-specific probeset mapping
Drosophila Orc2 subunit were added and incubated with (20.0.0) from the Molecular and Behavioral Neuroscience
the supernatant overnight. Sepharose beads were washed Institute (Brainarray) at the University of Michigan (Dai
three times with low-salt immunoprecipitation buffer et al. 2005) (brainarray.mbni.med.umich.edu/Brainarray/
and diluted in 10 ml of immunoprecipitation buffer. Sam- Database/CustomCDF). Array quality was assessed by com-
ples were boiled in loading buffer, separated in 10% SDS- puting relative log expression and normalized unscaled stan-
polyacrylamide gel, and transferred on Immobilon-P dard error, using the affyPLM package (version 1.46.0).
membrane (#IPVH00010; Millipore, Bedford, MA). The pres- Principal component analysis was performed using the
ence of GFP-Orc6 fused protein was detected with a mouse prcomp R function with expression values that had been nor-
anti-GFP (B2, #sc9996; Santa Cruz Biotechnology) anti- malized across all samples to a mean of zero and a standard
body. A mouse anti-Orc5 antibody was used to verify ORC deviation of one. Differential expression was assessed using
complex immunoprecipitation. the moderated (empirical Bayesian) t-test implemented in
Mitotic chromosome preparation the limma package (version 3.26.9) (i.e., creating simple lin-
ear models with lmFit, followed by empirical Bayesian adjust-
Preparation of mitotic nuclei was described previously ment with eBayes). Correction for multiple hypothesis testing
(Lebedeva et al. 2000). Briefly, third instar larval neural gan- was accomplished using the Benjamini–Hochberg false dis-
glia were incubated in 0.075M KCl for 5 min, fixed in meth- covery rate. Human homologs of fly genes were identified
anol with acetic acid (3:1) for 20 min, and then dispersed in a using HomoloGene (version 68). All microarray analyses
drop of 50% propionic acid on a slide. Then slides were dried were performed using the R environment for statistical com-
and stained with 5% Giemsa’s solution. puting (version 3.2.0). Gene ontology terms analysis was
conducted using DAVID Bioinformatics Resources 6.8
Western blot of larval brains
(david.ncifcrf.gov) (Huang et al. 2009).
Freshly dissected brains were incubated with 450 mM NaCl,
Data availability statement
0.5% NP-40 in PBS buffer for 1 hr, and samples were centri-
fuged 10 min at 10000 3 g. Pellets were washed three times Strains and plasmids are available upon request. The authors
with 100 mM NaCl, 0.5% NP-40 in PBS buffer and used for affirm that all data necessary for confirming the conclusions of

Humanized Drosophila Model of MGS 997

 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 do-
mains share 27% amino acid identity and 48% sim-
ilarity. 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 muta-
tion (Y225S) are indicated in red.

the article are present within the article, figures, and tables. lethality; however, the expression of Orc6-HD resulted in a
Supplemental material available at figshare: https://doi.org/ full rescue and obtained flies were undistinguishable from
10.25386/genetics.13070288. the flies rescued with wild-type Drosophila Orc6 (Orc6-
DmWT) (Figure 2A).
Orc6 is a subunit of the ORC complex. Therefore, we
Results
analyzed the ability of Orc6 proteins described above to
Establishing Drosophila strains to study the functions of associate with Drosophila ORC complex. We employed anti-
human Orc6 in vivo Orc2 antibodies for immunoprecipitation of ORC complex,
and antibodies against Orc5 subunit were used as a rigorous
The metazoan Orc6 consists of two domains (Figure 1):
control for the presence of the whole ORC complex in immu-
a larger N-terminal domain that carries a homology with
noprecipitated material. We found that Orc6-HD but not
TFIIB transcription factor and is important for DNA binding
Orc6-DH or wild-type human Orc6-HsWT immunoprecipi-
(Chesnokov et al. 2003; Balasov et al. 2007; Liu et al. 2011),
tated with the rest of the ORC complex (Figure 2B).
and a shorter C-terminal domain essential for the interaction
Furthermore, the MCM helicase association with the
of the protein with the core ORC through its Orc3 subunit
chromatin was diminished in Orc6-DH as compared to the
(Bleichert et al. 2013). The C-terminal domain of metazoan
Orc6-HD strain (Supplemental Material, Figure S1A), indi-
Orc6 is also important for the function of Orc6 in cytokinesis
cating that the pre-RC formation is impaired in the presence of
(Prasanth et al. 2002; Chesnokov et al. 2003; Huijbregts et al.
Orc6-DH form of the protein.
2009; Bernal and Venkitaraman 2011; Akhmetova et al.
Since Orc6-HD compensated for the orc6 deletion similar
2015).
to the wild-type Drosophila Orc6, this transgene can be used
In our previous work we generated a deletion of the orc6
for studying mutations in the human large N-terminal TFIIB-
gene in Drosophila allowing the studies of the protein in vivo.
like domain portion of Orc6 protein in the live animal model.
We showed that the orc6 deletion resulted in defects in DNA
replication as well as abnormal chromosome condensation The analysis of flies carrying MGS mutation localized in
and segregation (Balasov et al. 2009). Orc6 is the least con- the N-terminal domain of Orc6
served among ORC subunits, with metazoan Orc6 proteins In our previous analysis of Orc6 functions in vivo, we showed
showing significantly higher homology as compared to bud- that the mutation of the conserved tyrosine to serine (Y225S)
ding yeast protein. The full-length human orc6 gene (Orc6- in the C-terminal domain of Drosophila Orc6 resulted in third
HsWT) under control of native orc6 promoter did not rescue instar lethality associated with defects in DNA replication and
lethality of the Orc6 deficient flies (Balasov et al. 2009). chromosome abnormalities (Balasov et al. 2015). In humans,
However, human Orc6 protein was able to bind chromosomes the corresponding mutation (Y232S) is associated with MGS.
when expressed in Drosophila (Liu et al. 2011). We asked if The molecular analyses of the mutation in this Drosophila
the expression of a hybrid protein designed from the human model of MGS revealed that it disrupted the interaction be-
DNA binding domain of Orc6 and the Drosophila C terminus tween Orc6 and the rest of the ORC through the Orc3 subunit,
responsible for the interaction with a core ORC might rescue thereby impairing pre-RC formation and MCM recruitment,
the lethality associated with the Orc6 deletion. Two con- which is essential for DNA replication (Bleichert et al. 2013;
structs were created (Figure 2A). The first consisted of the Balasov et al. 2015).
Drosophila N-terminal TFIIB-like domain and human C ter- Recently, the first diagnosed MGS case in China was de-
minus (Orc6-DH); the second construct contained human scribed (Li et al. 2017). The patient had a short stature,
N-terminal domain and Drosophila C terminus (Orc6-HD). microtia, small patella, and craniofacial abnormalities char-
Both constructs were tested for their ability to rescue orc6 acteristic for MGS. It was reported that the patient carried a
deletion. The expression of Orc6-DH did not rescue the novel homozygous mutation in the orc6 gene, resulting in a

998 M. Balasov, K. Akhmetova, and I. Chesnokov

 Figure 2 Design of hybrid Orc6 and Orc6 mutants.
(A) Diagrams of transgenic constructs. Black repre-
sents 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) Immunopre-
cipitation of the ORC complex with anti-Orc2 anti-
bodies from ovaries expressing GFP-tagged Orc6
shown in A. (C) The position of the K23E substitu-
tion in Drosophila Orc6 (red). (D) Immunoprecipita-
tion of ORC complex using Orc2 antibodies from
Drosophila Orc6-K23E mutant fly ovaries. Immuno-
precipitations 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.

change of A to G at position 67, which corresponds to the As shown in Figure S3, mutant Orc6 protein carrying the
conversion of lysine at position 23 to glutamic acid (K23E). K23E mutation was able to pull down Orc3 in the in vitro
Interestingly, this mutation is localized within N-terminal transcription/translation reaction as well as the wild-type
TFIIB-like domain of the protein, unlike the earlier described Drosophila Orc6. We conclude that K23E mutation in Orc6
Y232S MGS mutation (Bicknell et al. 2011a; de Munnik et al. does not affect its association with the core ORC.
2012a), which effects the C-terminal domain of the protein Replication defects often result in a loss of chromosome
(Figure 1). integrity. Therefore, we investigated mitotic chromosomes in
We then set about determining the molecular conse- developing larval brains, since this tissue is known to provide
quences of the new MGS mutation. Since only wild-type the best mitotic figures. The normal Drosophila melanogaster
Drosophila Orc6 and hybrid Orc6-HD resulted in viable karyotype consists of four pairs of chromosomes (Figure 3A,
adults, we incorporated K23E mutation into Drosophila Orc6-DmWT). In larvae carrying orc6 deletion (Figure 3A,
Orc6 gene and human N-terminal domain of the Orc6-HD Orc6 deletion) or expressing Orc6-DH (Figure 3A, Orc6-
hybrid construct (Figure 2, C and E). The resulting con- DH) or Orc6-HsWT (Figure 3C, Orc6-HsWT) constructs on
structs, Orc6–DmK23E and Orc6-HDK23E, were introduced orc6 deletion background, chromosomes lost parts of their
into flies carrying the homozygous deletion of the orc6 gene. arms, appeared aberrantly condensed, and fragmented. On
Three independent transgenic fly stocks were set up for each the other hand, Orc6-HD transgene expression restored mi-
mutant construct (Figure S2). totic chromosome structure (Figure 3A, Orc6-HD). The in-
To analyze the ability of mutant proteins to interact with corporation of the K23E mutation into Orc6 resulted in
the rest of the complex, we immunoprecipitated ORC from chromosome defects and fragmentations (Figure 3, B and
obtained transgenic flies using Orc2 antibodies. Drosophila C). To quantify chromosome integrity, we counted percent-
Orc6 carrying Y225S mutation (Orc6-DmY225S) was used as ages of severely affected mitoses (as in Orc6 deletion mu-
a negative control for complex association since this mutation tant), mildly/moderately affected (as in Orc6-HDK23E-4),
abolished the interaction of Orc6 protein with ORC complex and normal mitoses (as in Orc6-DmWT) (Figure 3D). As
(Balasov et al. 2015). As expected, Orc6-DmY225S protein expected, two transgenic stocks, Orc6-DmWT and Orc6-
did not immunoprecipitate with ORC (Figure 2, D and F). HD, restored all karyotypes (100%) to the normal pattern.
However, both Drosophila and hybrid Human-Drosophila Orc6 deletion, Orc6-DH, and Orc6-HsWT contained .95% of
Orc6 proteins carrying the K23E substitution were present defective mitoses. Orc6-DmK23E and Orc6-HDK23E mutants
in the immunoprecipitated material indicating that the inter- all contained some defective mitoses with different degree of
action with core ORC was not affected by the mutation (Fig- severity (Figure 3D). The minor chromosome defects were
ure 2, D and F). It was previously shown that metazoan Orc6 also reported for the human patient carrying the K23E mu-
associated with ORC via its Orc3 subunit (Bleichert et al. tation (Li et al. 2017).
2013). Therefore, we tested whether K23E mutation would To investigate replication defects more rigorously, we an-
disrupt a direct interaction between Orc6 and Orc3 subunits. alyzed BrdU incorporation in larval brains of the fly stocks

Humanized Drosophila Model of MGS 999

 Figure 4 BrdU incorporation in larval brains. Brains of third instar larvae
expressing specified transgenes on orc6 deletion background were la-
Figure 3 Mitoses in larval neuroblasts. Brains of third instar larvae beled with BrdU. Transgenes are as follows: (A) wild-type Drosophila Orc6,
expressing specified transgenes on orc6 deletion background were ana- (B) Orc6-DH hybrid, (C) Orc6-HD hybrid, (D) wild-type human Orc6, (E)
lyzed for the karyotype. (A) Orc6 deletion mutant and hybrid Orc6 (Orc6- Drosophila Orc6-K23E mutant, and (F) Orc6-HD hybrid with the K23E muta-
DH, Orc6-HD) compared to Drosophila wild type (Orc6-DmWT). (B) Three in- tion. Bars represents 50 mm. (G) Percentages of BrdU-labeled area relative to
dependent DmOrc6 transgenics bearing the K23E mutation (Orc6-DmK23E- total brain area (n = 5). * indicates a statistically significant difference
3, Orc6-DmK23E-4, Orc6-DmK23E-6). (C) Three independent hybrid compared to Orc6-DmWT; ns indicates no significant difference.
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 mm. (D) Percentages of defective
Minor defects (missing scutellar bristles) appeared in 18% of
(orange and red) and normal (green) karyotypes. Total number of mitoses
analyzed is shown above the chart. 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
expressing hybrid or mutant proteins. Brains were underde- (Figure S2 and Table 1). Another stock Orc6-DmK23E-6 pro-
veloped and BrdU incorporation was reduced in flies carrying duced very few (3%) homozygous adults (Table 1). All of the
the Orc6-HsWT or Orc6-DH constructs (Figure 4, B and4D). No surviving flies had defective scutellar bristles and reduced
difference was observed between the Orc6-DmWT and Orc6- eye size (Figure 5C). The eye of Drosophila is a well-estab-
HD hybrid constructs (Figure 4, A and C). The larval brains of lished model to study proliferation defects. The compound
the fly stocks expressing mutant proteins Orc6-DmK23E (Figure eye of Drosophila consists of uniform regular facets, and the
4E) or Orc6-HDK23E (Figure 4F) were also underdeveloped final number of them is determined during several mitotic
and showed reduced BrdU labeling. Quantifications confirmed divisions in third instar larvae. Any delay or disturbance dur-
that Orc6-HD was not statistically different from Orc6-DmWT, ing the course of divisions result in reduced size and abnor-
whereas all other analyzed mutants were defective (Figure 4G). mal shape of the adult eye. This phenotype was consistent
Next, we calculated the percentage of homozygous flies with a disrupted Orc6 function in replication and cell cycle
rescued with different transgenes driven by native Orc6 progression. Another interesting observed phenotype was the
promoter (Table 1). We found that stock Orc6-DmK23E-4 inability of mutant flies to fly. Rescued adult flies carrying
resulted in 28% of adult viability. The majority of rescued Orc6-DmK23E could not fly but were able to jump and walk.
flies (82%) were indistinguishable from wild type (Figure 5A). Previously we reported that flies with C-terminal Orc6 MGS

1000 M. Balasov, K. Akhmetova, and I. Chesnokov

Table 1 Rescue of the orc6 deletion with the expression of different Orc6 transgenes under control of native or tubulin promoter
Orc6-Dm Orc6-Dm Orc6-Dm Orc6-Dm Orc6-HD Orc6-HD Orc6-HD Orc6-Hs Orc6-Hs
WT K23E-3 K23E-4 K23E-6 Orc6-DH Orc6-HD K23E-4 K23E-6 K23E-9 WT K23E

Native promoter 1 – 1 1/- – 1 – – – – –

80% 0% 28% 3% 0% 65% 0% 0% 0% 0% 0%
(495) (411) (422) (276) (678) (683) (393) (301) (296) (351) (413)
Tubulin promoter 1 1 1 1 1 1 1 1 1 1 –
91% 39% 54% 48% 20% 80% 74% 79% 33% 27% 0%
(596) (622) (427) (496) (790) (495) (591) (569) (670) (774) (315)
Percentages of rescued flies were calculated based on expected segregation of phenotypes. Total progeny analyzed is shown in parentheses. (+), the expression of transgene
restores viability of orc6‐deleted flies to adult stage; (2), no rescue, (6) very low rescue percentage.

mutation (Y225S) also missed scutellar bristles and were binding sites for Orc6 within the ori-b fragments. However,
flightless (Balasov et al. 2015). The observed bristle defect the mobility of DNA-protein complex changed significantly
and flightless phenotype could be a common MGS feature in when Orc6-K23E was used in the reaction. The mutant com-
Drosophila. plex migrated faster compared to the wild-type protein, prob-
In our previous study describing our fly model of MGS ably due to a reduced template occupancy resulting from the
based on Orc6 Y225S mutation (Balasov et al. 2015), we K23E mutation. Increasing the concentration of the mutant
showed that the elevated expression of Orc6 Y225S trans- protein in electrophoretic mobility shift assay (EMSA) reac-
gene rescued the orc6 deletion phenotype. We investigated tions slightly improved the DNA binding of Orc6-K23E pro-
whether it would be the case for K23E mutation. Similar tein (Figure S4B).
to the Y225S Orc6 mutant, all K23E orc6 transgenes have When the K23E substitution was made in the human Orc6
an UAS promoter upstream of native orc6 promoter. The protein or in a hybrid protein Orc6-HD containing human
UAS promoter allows increased Orc6 transgene expression N-terminal TFIIB-like domain, the overall DNA binding ability
by crossing to flies bearing the tubulin promoter-driven of the resulting mutant proteins was significantly diminished
GAL4 (see Materials and Methods). As summarized in Ta- (Figure 6, B and C). We would like to point out that the DNA
ble 1, elevated expression of both Drosophila and human- binding pattern for human Orc6 is different from Drosophila
Drosophila hybrid transgenes carrying the K23E mutation protein, with the majority of the DNA-protein complex mi-
promoted flies to adult stage. Remarkably, these rescued grating at the top of the EMSA gel. This difference between
flies also had defects of the scutellar bristles and were not fly and human Orc6 binding to DNA was reported previously
able to fly, similar to the phenotypes observed in the fly (Balasov et al. 2007; Liu et al. 2011).
model of MGS based on the Orc6 Y225S mutation (Balasov To visualize the Orc6 mutants binding in vivo, we used
et al. 2015). GFP-Orc6 bearing fly stocks described in Materials and
Overall, we concluded that both MGS mutations resulted Methods. The UAS promoter in the construct allows for
in reduced DNA replication and chromosome defects, mani- GAL4-induced expression using the GAL4/UAS binary sys-
festing in similar phenotypes; however, the molecular mech- tem (Brand and Perrimon 1993; Duffy 2002). GFP-Orc6
anisms driving this phenotype might be different. The Y225S expression was induced in salivary glands of third instar
substitution impedes Orc6 binding to the ORC complex larvae to test chromosome binding of Orc6 mutants. Nuclei
(Bleichert et al. 2013; Balasov et al. 2015). The K23E sub- of Drosophila salivary glands contain polytene chromo-
stitution resides in the N-terminal domain of Orc6 that is somes that can be easily visualized with microscopy. Both
important for DNA binding. Therefore, we investigated the Gal4-induced Orc6-DmWT and Orc6-DmK23E (Figure 6D)
ability of Drosophila, human, and hybrid Orc6 proteins car- were found to be associated with polytene chromosomes. In
rying the K23E mutation to bind DNA. contrast, over-expressed Orc6-HDK23E and Orc6-HsK23E
mutants showed significantly reduced DNA binding and as-
K23E mutations disrupt DNA binding ability of
Orc6 protein sociation with chromosomes (Figure 6, E and F), in agree-
ment with the EMSA DNA binding experiments shown in
Six recombinant proteins were purified using an E. coli Figure 6, B and C. Together, our data on DNA binding sug-
expression system: Drosophila (Orc6-DmWT and Orc6-DmK23E), gest that K23E mutation has a debilitating effect on DNA
hybrid human/Drosophila (Orc6-HD and Orc6-HDK23E), binding ability of Orc6.
and human (Orc6-HsWT and Orc6-HsK23E) (Figure S4A).
K23E mutation results in the instability of the
As shown in Figure 6A, both Drosophila Orc6 wild-type
Drosophila, but not human, Orc6
protein and Orc6 carrying the K23E mutation were able to
bind in vitro with the ori-b DNA fragment derived from the Often mutations lead to a protein unfolding that may affect its
origin of DNA replication associated with a chorion gene functions, such as DNA binding. Therefore, we tested the
cluster. Binding was very tight, with the majority of the la- effect of the K23E substitution on protein stability. In this
beled probe bound by the protein, as we observed before experiment the unfolding of the protein was studied by
(Balasov et al. 2007), suggesting the presence of multiple measuring the intrinsic fluorescence intensity at a wavelength

Humanized Drosophila Model of MGS 1001

 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.
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 expres-
sion of mutant protein carrying Y225S substitution rescued
lethality, restored normal karyotype, and allowed detec-
tion 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
Figure 5 Phenotypes of orc6 deletion flies rescued with Orc6-DmK23E. with Drosophila orc6 gene deletion (Table 1). We asked if
(A) Flies expressing wild-type Drosophila Orc6. (B) Orc6DmK23E-4 mutant
flies have mild phenotype: eye size and facets number are normal; how-
overexpression of the human Orc6 would rescue orc6 defi-
ever, 18% of flies have missing or defective scutellar bristles (arrows). (C) ciency in Drosophila. Using a GAL4/UAS binary system, we
All surviving flies carrying Orc6DmK23E-6 showed reduced eye facets boosted the expression of human wild type and K23E mutant
number and multiple defects of scutellar bristles (arrows). in flies. We found that the elevated expression of the wild-
type human Orc6 was able to rescue flies to viability (Table 1)
of excitation corresponding to tryptophan. Tryptophan fluo- with restoration of normal karyotype (Figure 8A). We also
rescence is very sensitive to protein conformational changes observed human Orc6 with other ORC subunits after immu-
and thus provides information about changes in secondary noprecipitation with anti-Orc2 antibodies (Figure 8B). Re-
and/or tertiary structure. A decrease in fluorescence intensity markably, rescued adult flies displayed an upheld wing
and shift in the maximal emission wavelength reflect protein phenotype (Figure 8, C and D) and were not able to fly (File
unfolding (Royer 2006). We measured the tryptophan fluo- S1, movie file), similar to the Drosophila C-terminal MGS
rescence of Drosophila and human Orc6 proteins at the in- mutation Y225S flies (Balasov et al. 2015). However, when
creasing temperatures and compared them with mutants K23E mutation was introduced into the human Orc6, the
carrying the K23E mutation. As presented in Figure 7A, the resulting mutant Orc6-HsK23E transgene was not able to res-
K23E mutation in human Orc6 did not change a stability of cue orc6 deficient flies to viability even under overexpressed
the mutant protein relative to wild type at all tested temper- conditions (Table 1), and no improvements were observed
atures; however, the Drosophila Orc6 carrying the K23E mu- during development of the flies (Figure 8A, lower row). The
tation was more susceptible to the heat and began unfolding loose association of human Orc6 with the core complex some-
at temperatures over 35°, suggesting that the amino acid what mimics the disruptive effect of Y225S substitution in
substitution causes an instability in fly protein. Importantly, Drosophila Orc6 for interaction with ORC. As a result, the
fluorescence intensity of the Drosophila mutant relative to Orc6-HsK23E mutant appears to be defective in both DNA
wild type was significantly lower even at 25° (Figure 7B). binding (Figure 6, C and F) and in interacting with the core
This indicates that some part of the mutant protein is ORC, therefore it was not possible to rescue a lethality re-
already unfolded at temperatures optimal for Drosophila gardless of the protein expression level (Table 1).
development. We hypothesized that elevated temperature
Microarray analysis of the MGS mutation in Drosophila
may increase protein unfolding and further affect the DNA
binding ability of Drosophila Orc6. To test this hypothesis we In our previous study, we found that flies carrying Y225S
repeated EMSA with wild-type and K23E mutant protein mutation had rough spots in the eye, irregular hair patterns,
probes heat shocked at 50° for 15 min. Mutant Orc6 did and missing bristles—features possibly associated with minor

1002 M. Balasov, K. Akhmetova, and I. Chesnokov

 Figure 6 DNA and chromosome
binding ability of hybrid and mu-
tant 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 pro-
teins; 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 com-
petitor poly(dI-dC) and poly(dG-dC)
is shown in nanograms. Polytene
chromosomes isolated from sali-
vary glands expressing GFP-tagged
Orc6 were immunostained with
anti-GFP antibodies, and DNA was
stained with DAPI.

cell polarity defects (Balasov et al. 2015). These phenotypes upregulated more than two times. This result is consistent
could indicate potential role of Orc6 in other processes or with a relatively mild phenotype associated with K23E mu-
pathways apart from replication. To investigate this possibil- tation compared to Y225S mutation. Next, we filtered up-
ity further, we conducted a microarray analysis of gene ex- and downregulated genes in both mutants for common
pression to identify the genes/pathways that are affected by genes. The end results consisted of two short lists of 9 down-
the MGS mutations. Orc6 carrying Drosophila Y225S MGS regulated genes and 14 upregulated genes (Table S1). We
mutation rescued adult flies only when overexpressed, there- analyzed the expression patterns of genes based on modeEN-
fore orc635/orc635; Orc6-DmY225S/tubP-GAL4 adults were CODE RNA-seq data (flybase.org) and found that all nine
subjected to the microarray analysis against orc635/orc635; downregulated genes had maximum expression level in ova-
Orc6-DmWT/tubP-GAL4 adults. From the total analyzed ries. The ovary is the organ where the majority of the repli-
13557 genes, 1446 genes were downregulated more than cation events happens in adult flies, and so they are more
two times and 1363 genes were upregulated more than sensitive to replication defects. The 14 upregulated genes
two times. Gene ontology analysis revealed downregulated showed high expression in head and digestive system. Most
DNA replication, DNA repair, and cell proliferation processes of them encoded products with endopeptidase and ubiquitin-
in the top 10 scores (Table S1). One of the Orc6-DmK23E transferase activities. We hypothesize that the elevated ex-
MGS mutant transgenes rescued flies to the viability without pression of these genes could reflect degradation/utilization
overexpression; therefore, orc635/orc635; Orc6-DmK23E-4/ processes in tissues where proliferation defects resulted in
Orc6-DmK23E-4 were analyzed and compared to orc635/ cell damage. Overall microarray analysis did not reveal any
orc635; Orc6-DmWT/Orc6-DmWT. In this case, only 33 genes additional genes/pathways affected by MGS mutations with
were downregulated more than two times and 25 genes were the exception of replication and proliferation. It should be

Humanized Drosophila Model of MGS 1003

 microscopy revealed that all large muscle fiber groups were
present in MGS mutants (Figure S5). An inability to fly
might also result from defects in IFM innervation. To test
this, we induced RNA interference of orc6 specifically in
motor neurons. Remarkably, reduction of orc6 expression
in motor neurons also caused an inability to fly (data not
shown). This experiment suggests that Orc6 might partici-
pate in motor neuron development during metamorphosis.

Discussion
MGS is a rare human disease associated with microcephaly,
short stature, and multiple developmental defects (Bicknell
et al. 2011a; de Munnik et al. 2012a,b; Kerzendorfer et al.
2013). Mutations in a number of factors involved in DNA
replication have been found to be causative for this disease
(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). Several mutations causing MGS were found in ORC
subunits, including Orc6 (Bicknell et al. 2011a,b; Guernsey
et al. 2011). In humans, Y232S substitution in Orc6 corre-
Figure 7 Tryptophan fluorescence of Drosophila and human Orc6 pro-
sponds to a mild clinical appearance of MGS (Bicknell et al.
teins. (A) Emission maximum wavelengths at gradually increasing temper-
atures are shown. Shift in the maximal emission wavelength reflects 2011a; de Munnik et al. 2012a); however, the corresponding
protein unfolding. (B) Absolute intensity at each temperature point. mutation in Drosophila (Y225S) is lethal and molecular and
DmWT and DmK23E indicate Drosophila Orc6 wild type and Orc6-K23E cell analysis revealed significantly reduced DNA replication
mutant, respectively, and HsWT and HsK23E indicate human Orc6 wild and chromosome fragmentation in cells and tissues (Balasov
type and Orc6 HsK23E mutant, respectively.
et al. 2015). Importantly, flies carrying the Orc6-Y225S mu-
tation can be rescued by the elevated expression of the trans-
gene (Balasov et al. 2015).
noted that RNA was isolated from the whole body of the adult The second known mutation in human Orc6 related to
flies, which might obscure the difference between specific MGS is a homozygous deleterious mutation within the gene.
tissue and developmental stages. For a more rigorous conclu- Small deletion c.602-605delAGAA generated stop codon after
sion, tissue-/stage-specific microarrays should be performed. 202 codons (Shalev et al. 2015). The severity of abnormal
embryological development in humans coincided with a le-
Analysis of the flightless phenotype
thal embryonic phenotype in Drosophila for Orc6 having only
The flightless phenotype is a characteristic of all analyzed first 200 amino acids (Balasov et al. 2009). The elevated
Orc6-based MGS mutants in Drosophila, including previously expression of this mutant protein did not rescue a lethal phe-
described Y225S (Balasov et al. 2015) and a new K23E mu- notype of flies (Balasov et al. 2015).
tations. In our current study, GAL4 . UAS overexpression A third known mutation in human Orc6 related to MGS was
of Drosophila K23E mutant rescued the lethality and bristle recently reported as the mutation responsible for the K23E
defects but did not restore the ability to fly. We assayed flight substitution (Li et al. 2017). This mutation caused a pheno-
performance by dropping flies individually into a 90-cm type in Drosophila that varies in severity from lethality to
cylinder. Flies expressing wild-type Drosophila Orc6 immedi- relatively normal adults. This newly described K23E muta-
ately escaped the cylinder or landed on the wall and flew tion localizes in the DNA binding N terminus of the protein.
away in seconds. In contrast, mutants landed on the bottom Not surprisingly, biochemical and cytological analysis revealed
of the cylinder, they did not try to escape and were easy to that K23E mutation in both Drosophila and human Orc6 re-
collect. Interestingly, the flies rescued with human Orc6 were duced DNA binding in gel-shift experiments. In vivo, this mu-
not able to fly either (File S1, movie file). tation leads to the inability of Orc6-HsK23E or Orc6-HDK23E
To understand the cellular mechanism of flightless phe- to bind with chromosomes, but the chromosome association
notype, we performed histological analyses of the major of Orc6-DmK23E is not significantly affected. This observa-
thoracic muscles. In Drosophila, large indirect flight muscles tion coincided with the survival rates as flies carrying Orc6-
(IFMs) mediate flight by contraction and expansion of the DmK23E often were able to progress to the adulthood.
thoracic cuticle. IFMs are composed of six dorsal longitudi- Interestingly, K23E mutation leads to the instability of the
nal muscles and seven dorsoventral muscles (Bernard et al. fly protein, with a difference already noticeable at 37°. However,
2003). Therefore, we analyzed histological transverse sec- in the case of human protein, the same mutation did not
tions of IFMs for signs of degeneration or atrophy. Light change protein conformation compared to the wild type,

1004 M. Balasov, K. Akhmetova, and I. Chesnokov

 Figure 8 Rescue of orc6 deletion flies with human
Orc6. (A) Mitoses in neuroblasts of orc6 deletion
mutant rescued with human Orc6 wild type or with
the K23E MGS mutation. Expression of transgenes
was driven with either native orc6 promoter (np .)
or with strong constitutive tubulin promoter (tub .).
(B) Immunoprecipitation of the ORC complex with
anti-Orc2 antibodies from ovaries expressing
GFP-tagged Orc6 transgenes. In1 and In2 indicate
1/10 and 1/50 of total immunoprecipitation reaction,
respectively. (C) Adult flies rescued with wild-type
Drosophila Orc6. (D) Adult flies rescued with tubulin
promoter driven overexpression of wild-type human
Orc6.

even at the higher temperatures. Normal body temperature Orc6 protein containing human N-terminal domain and
for humans, 37°, is lethal for Drosophila, suggesting that evo- Drosophila C terminus. This transgene rescued orc6 deletion
lutionary changes occurred in warm-blooded animals to flies to viability and they were indistinguishable from the
withstand thermal challenges otherwise lethal for insects. wild-type animals. This indicates that the N-terminal TFIIB-
In summary, the two known MGS substitution mutations in like domain of both proteins involved in the same con-
Orc6 affect different functional domains of the protein and served functions between organisms. On the other hand,
result in either impaired DNA binding (K23E) by Orc6 or a the C terminus of both Drosophila and human Orc6 contains
loss of the protein association with the core ORC (Y232S). conserved motifs responsible for association with ORC com-
The consequences of both mutations include the reduced plex. Both flies and humans with C-terminal truncations do
amounts of hexameric ORC on DNA, an impaired pre-RC not survive to the adult stage (Balasov et al. 2009; Shalev
formation, and fewer origin firings. This in turn leads to the et al. 2015). However, the point mutation (Y232S) in human
replication, proliferation, and development defects manifest- Orc6 manifests in a mild postnatal phenotype (Bicknell et al.
ing in similar clinical features in both cases. 2011a; de Munnik et al. 2012a), while the corresponding
Drosophila and human Orc6 proteins have only 28% of Drosophila Y225S MGS mutant shows no difference from
sequence identity but are structurally similar and critical the lethal orc6 deletion. It is known that human Orc6 is
for the initiation of DNA replication (Duncker et al. 2009). loosely associated with the core ORC (Vashee et al. 2001;
The metazoan Orc6 consists of two domains. The larger Vashee et al. 2003; Ranjan and Gossen 2006), whereas
N-terminal domain (200 amino acids) carries a homology Drosophila Orc6 associates with other ORC subunits signifi-
with the transcription factor TFIIB and is important for DNA cantly more tightly (Gossen et al. 1995; Chesnokov et al.
binding (Chesnokov et al. 2003; Balasov et al. 2007; Liu et al. 1999; Chesnokov et al. 2001). This might explain why the
2011). The shorter C-terminal domain is important for the Y225S MGS mutation has a more dramatic effect on survival
function of Orc6 in cytokinesis (Prasanth et al. 2002; in Drosophila than the corresponding MGS (Y232S) mutation
Chesnokov et al. 2003; Huijbregts et al. 2009; Bernal and in humans.
Venkitaraman 2011; Akhmetova et al. 2015), as well as for In our earlier study (Balasov et al. 2015) we found that an
the interaction of Orc6 with core ORC (Bleichert et al. 2013). elevated expression of the fly protein carrying the Y225S
Full-length human Orc6 could not rescue a lethality associ- mutation rescued third instar lethality, restored normal kar-
ated with the loss of the fly gene when expressed under yotype, and the mutant protein was detected on DNA with
native orc6 promoter. Therefore, we created the hybrid the rest of ORC complex. Similarly, here we found that the

Humanized Drosophila Model of MGS 1005

elevated expression of human Orc6 allowed the formation of conserved C-terminal domain of Orc6. Am. J. Med. Genet. A.
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Bell, S. P., 2002 The origin recognition complex: from simple
carrying orc6 deletion. In some way human Orc6 in fly system
origins to complex functions. Genes Dev. 16: 659–672.
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Drosophila Orc6. In both cases the interaction of the proteins Bell, S. P., and B. Stillman, 1992 ATP-dependent recognition of
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Bernal, J. A., and A. R. Venkitaraman, 2011 A vertebrate N-end
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containing intact human N-terminal TFIIB-like domain 10.1083/jcb.201008125
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avenue to study new Orc6 mutations for medical and general origin recognition complex, cause microcephalic primordial
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the functions of human protein in a convenient heterologous a means of altering cell fates and generating dominant pheno-
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Acknowledgments Chen, S., M. A. de Vries, and S. P. Bell, 2007 Orc6 is required for
dynamic recruitment of Cdt1 during repeated Mcm2–7 load-
We thank Adam Gower from the Boston University Micro- ing. Genes Dev. 21: 2897–2907. https://doi.org/10.1101/gad.
array and Sequencing Resource Core Facility. We also thank 1596807
Chesnokov, I. N., 2007 Multiple functions of the origin recogni-
Dr. Peter Detloff for the critical reading of the manuscript.
tion complex. Int. Rev. Cytol. 256: 69–109. https://doi.org/
All research materials and data from our studies will be 10.1016/S0074-7696(07)56003-1
freely available to other investigators. This work was sup- Chesnokov, I., M. Gossen, D. Remus, and M. Botchan, 1999 As-
ported by a grant from the National Institute of General sembly of functionally active Drosophila origin recognition com-
Medical Sciences (GM121449 to I.C.). plex from recombinant proteins. Genes Dev. 13: 1289–1296.
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Chesnokov, I., D. Remus, and M. Botchan, 2001 Functional anal-
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Humanized Drosophila Model of MGS 1007