Supplementary information
Manuscript MMI-2015-15005
"Regulation of Plasmodium falciparum Origin Recognition Complex subunit 1 (PfORC1)
function through phosphorylation mediated by CDK like kinase PK5" by Deskmukh A and
Agarwal M et al.
Supplementary data: Materials and methods.
Purification of recombinant proteins.
For Ni-nitrilotriacetic acid (Ni-NTA) purification of His6-PfORC1N1-238 and PfPK5-His6
proteins, the bacterial pellet was lysed in lysis buffer (100 mM Na2HPO4, 100mM NaH2PO4,
50 mM Tris-HCl pH8.0, 300 mM NaCl, 1 mg/ml lysozyme, 10 mM β-mercaptoethanol and 100
μM PMSF) containing 10 mM imidazole. The lysate was cleared by centrifugation at 10000xg.
The cleared lysate was incubated with Ni-NTA beads for 1 hour at 40C. The beads were washed
in wash buffer and eluted with lysis buffer containing 500 mM imidazole. All the proteins were
dialyzed and stored at -800C.
The human cyclin/CDK complex proteins were purified by mixing the soluble fraction of the
bacterial lysate containing GST-cyclinA/GST-cyclinE and His6-CDK2 (Wohlschlegel et al.,
2001). The corresponding ORFs of cyclins and CDK2 were cloned in pGex6p2 and pET28a
respectively. The mixed soluble fraction was incubated at 4ºC for 1 hour with gentle mixing
followed by affinity chromatography purification using Ni-NTA beads. The purification profile
of co-purified cyclin/CDK2 complex was confirmed by loading it on 10% SDS-PAGE, which
subsequently was stained with Coomassie stain. The co-purified cyclin/CDK2 proteins were
dialysed and stored at -800C.
For the purification of GST fusion proteins (ORC11-164, ORC116-164, ORC125-164, ORC1N1164T2AS20A,
GST, PfCrk-5, Pfcyc1, Pfcyc4, ORC11-238, ORC125-238, ORC1382-689 and ORC1690-1189)
bacterial pellet was lysed in lysis buffer containing 1X PBS, 10 mM DTT, 2 mM EDTA, 1mg/ml
lysozyme and 100 µM PMSF. The lysate was cleared by centrifugation at 10000xg. The cleared
lysate was incubated with equilibrated glutathione sepharose 4B beads for 1 hour at 40C. The
beads were washed in wash buffer containing 300 mM NaCl and eluted with lysis buffer
containing 10 mM reduced glutathione. All the proteins were dialyzed and stored at -800C.
For purification of MBP fusion protein Ringo) and MBP alone, the bacterial pellet was lysed in
the lysis buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mg/ml lysozyme, 1 mM EDTA, 100
μM PMSF). The lysates were cleared by centrifugation at 10000xg. The soluble fraction was
incubated with Amylose resin beads at 40C for 1 hour. Beads were washed with lysis buffer
containing 300 mM NaCl and eluted with elution buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl
and 10 mM maltose).
Supplementary data: Figure legends
Figure S1.
Schematic diagram of PfORC1 with putative motifs. The canonical CDK phosphorylation
motif is [ST]PX[RK]. This represents the most salient features of CDK phosphorylation site
composition that contributes to the catalytic efficiency of phosphorylation. We searched for
similar type of linear motif in the PfORC1 by manually and using computational tools such as
PROSITE and ELM and found two such consensus sequences (TPKK and SPTK ) at the
extreme end of N-terminus of PfORC1 (Chang et al., 2007).
All known Anaphase Promoting Complex (APC, ubiquitin ligase) substrates contain a
destruction (D) box, composed of the sequence R-X-X-L-X-X-X-X-N (Glotzer et al., 1991) and
KEN box sequences. We searched for such sequences in PfORC1 and found same consensus D
box and KEN box sequences suggesting possible degradation of ORC1 protein through ubiquitin
pathway.
The presence of all the putative motifs (as described above) in PfORC1 along with their amino
acid co-ordinates have been indicated in the figure.
Figure S2.
hcycA/Cdk2 phosphorylate ORC11-164. (A) Under the similar experimental conditions,
hcycA/Cdk2 phosphorylate ORC11-164 robustly but not the ORC11-164T2AS20A. Coomassie stained
gel shows equal loading of wild type and mutant proteins. (B) ORC11-164 showed increased
phosphorylation in a dose dependent manner with same concentrations of hcycA/Cdk2. 200 ng to
2 µg of ORC11-164 protein were used. The Coomassie-stained gel shows increasing concentration
of GST-ORC11-164. C. In a separate experiment, concentration dependent kinase activity of
hcycA/Cdk2 was evaluated by keeping the same concentration of ORC11-164. Coomassie stained
gel shows equal loading of GST-ORC11-164.
Figure S3.
Proteins used in kinase assay. Coomassie gel shows protein profiles of different cyclins (GSTPfcyc1, and GST-Pfcyc4), kinases (PfPK5-His6 and GST-PfCrk-5), human cell cycle protein
(MBP-Ringo) and deletion mutants of ORC1 (GST-PfORC11-164 and GST-ORC125-164).
Figure S4.
Immunodepletion of PfPK5 from parasite lysate using antibodies against PfPK5. Schizont
stage parasite (~ 40 hours) lysate was subjected to immunodepletion either using antibodies
against PfPK5 or pre-immune sera followed by western blot analysis. The results indicate the
presence of a prominent band corresponding to PfPK5 in the pre-immune sera depleted lysate
while the band intensity is drastically reduced in the immune sera depleted lysate suggesting
successful immunodepletion. PfActin was used as loading control.
Figure S5.
PCR amplification of TARE-1, -2, -3 and upsE regions. PCR amplification using primer sets
specific for TARE-1, -2, -3 and upsE regions as detailed in S1 Table. Standard molecular mass
markers (kb) are shown on the left. The results show single PCR amplification product for the
respective primers.
Figure S6
(A) The association of Histone H3 at the TAREs and promoter of var genes remained unaltered
in the ring (~20 hours) and schizont (~ 40 hours) stages. ChIP reactions were performed by
immunoprecipitation of chromatin fraction from the respective stages using antibodies against
Histone H3 followed by real time PCR using primer sets from the different TAREs and var
promoter regions (as indicated at the bottom). Input from the respective stage was used for
normalization of PCR. Enrichment was calculated as percent input (ChIP/Input) using the Ct
value of the “Input” and “ChIP” after subtracting the corresponding Ct value of Pre-immune.
(B) PfORC1 is loaded at the TARE region but not at the control HRP region in a stage specific
manner. ChIP reactions were performed by immunoprecipitation of chromatin fraction obtained
from ring (~20 hours) and schizont (~40 hours) stage parasites using antibodies against PfORC1
and followed by real time PCR using primer sets from the TARE-2 and control HRP (Histidine
Rich Protein) locus (Deshmukh et al., 2012). Input of the respective stage was used for
normalization of PCR. PfORC1 showed robust binding at the TARE-2 region but not at the
control HRP region during the ring stage. The binding of PfORC1 was drastically reduced at the
TARE-2 region during schizont stage. Enrichment was calculated as percent input (ChIP/Input)
using the Ct value of the “Input” and “ChIP” after subtracting the corresponding Ct value of Preimmune.
Figure S7.
Subcellular localization of ORC1N1-238T2AS20A-GFP and endogenous ORC1 in the schizonts.
Parasite lysate from schizont stage (~ 40 hours) was fractionated into cytoplasmic (CF) and
nuclear (NF) fractions and analysed by western blot analysis using polyclonal antibodies raised
against GFP or PfORC1. ORC1N1-238T2AS20A was found exclusively in the nuclear fraction
whereas endogenous ORC1 was found mostly in the cytoplasmic fraction. Aldolase (a
cytoplasmic protein) and Histone 3 (a nuclear protein) were used as markers for fractionation.
The molecular mass markers were shown on the left.
Table S1 List of primers used in this study.
A. Primers used for gene expression
(1.)
(2.)
(3.)
(4.)
(5.)
(6.)
(7.)
(8.)
(9.)
(10.)
(11.)
ORC1-1BamHIF
ORC1-46BamHIF
ORC1-75BamHIF
ORC1-492XhoIR
ORC1-714XhoIR
ORC1-1144BamHIF
ORC1-2067XhoIR
ORC1-2068BamHIR
ORC1-3567XhoIR
PK5-1NheIF
PK5-864XhoIR
5’-CGGGATCCATGACTCCTAAGAAAAAAATATT-3’
5’-CGGGATCCAATGAAATATTAAGTCCAACAAAAA-3’
5’-CGGGATCCATAAAATTAAATGTTAGTAAATTAAATA-3’
5’-CCCTCGAGTCATTCATCCAAGGAATTTGATAATG-3’
5’-CCCTCGAGTCACATTTTTTGTTGACATGTATTAT-3’
5’-CGGGATCCCCTAGTGAAAATTACAAAAAAC-3’
5’-CCCTCGAGTCATTTATTATTTTTTTTTTGGTTCGC-3’
5’-CGGGATCCAAGGAATATATCAATAAAGCTCA-3’
5’-CCGCTCGAGGTAAAAGTTTAATTTCTTTGAAC-3’
5’-CGGCTAGCATGGAGAAATATCATGGTTTAG-3’
5’-CCCTCGAGATTGTTTTCTTTAAAATACGCATG-3’
Note: Number before restriction site denotes nucleotide coordinate in the gene ORF. Restriction
site sequence is underlined.
The prime numbers are mentioned in the parenthesis for the respective gene.
ORC11-164 (1,4), ORC116-164 (2,4) ORC125-164 (3,4), ORC11-238 (1,5), ORC125-238 (3,5), ORC1382689(6,7), ORC1690-1189 (8,9) and PK5(10,11).
B. Primers used for GFP constructs
ORC1-1FKpnI
ORC1-700RAvrII
5’-CGGGGTACCATGACTCCTAAGAAAAAAATATT-3’
5’-TGGACCTAGGCATTTTTTGTTGACATGTATTAT-3’
Note: Number before restriction site denotes nucleotide coordinate in the gene ORF. Restriction
site sequence is underlined.
C. Primers used for site directed mutagenesis
ORC1-T2AF
ORC1-T2AR
ORC1-S20AF
ORC1-S20AR
5’-ATGGCTCCTAAGAAAAAAATATTTCAA-3’
5’-TTGAAATATTTTTTTCTTAGGAGCCAT-3’
5’-GATAATGAAATATTAGCTCCAACAAAAAAAGGG-3’
5’- CCCTTTTTTTGTTGGAGCTAATATTTCATTATC-3’
D. Primers used for EMSA
TelomereF
TelomereR
5’-AATCCGTCGAGCACATGTTT-3’
5’-CAGAATTCGCTTGGAATTCC-3’
(Mancio-Silva et al., 2008)
E. Primers used for ChIPqPCR
Primer name
TARE1-887F
TARE1-1033R
TARE2-4195F
TARE2-4345R
TARE3-6224F
TARE3-6349R
UpsE-57322F
UpsE-57475R
Primer sequence
5’-GTTCTTGACTTAACTTTTTATTCTTAC-3’
5’-GAAGAAAGTAAGAGTAAGGACCTAG-3’
5’-GATGTTGTAGATGATGTCGTTT-3’
5’-ACCTCCATGCATATAAGTAAAC-3’
5’-CGTCAACAAAATATGGTACAC-3’
5’-ATGAAAAATGGGTTGCAC-3’
5’-ATCACCATAATTAATATGAGAATCA-3’
5’-AATAATAGATAGCTATTACAAGAAATATG-3’
Genomic location
Chromosome 12
Chromosome 12
Chromosome 12
Chromosome 12
Chromosome 12
Chromosome 12
Chromosome 12
Chromosome 12
Note: Number in the primer name denotes nucleotide coordinate on the chromosome.
References:
Chang, J.E., Begum, R., Chait, B.T., Gaasterland. T. (2007) Prediction of Cyclin-Dependent
Kinase Phosphorylation Substrates. PLoS ONE 10: 1371.
Deshmukh, A.S., Srivastava, S., Herrmann, S., Gupta, A., Mitra, P., Gilberger, T.W., and Dhar,
S.K. The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var
gene silencing. Nucleic Acids Res 40: 5313-5331.
Glotzer. M., Murray, A.W., Kirschner, M. W. (1991) Cyclin is degraded by the ubiquitin
pathway. Nature 349, 132-137.
Wohlschlegel, J. A., Dwyer, B. T., Takeda, D. Y., and Dutta, A. (2001). Mutational analysis of
the Cy motif from p21 reveals sequence degeneracy and specificity for different cyclindependent kinases. Mol Cell Biol 21, 4868-4874.
Supplementary figures:
Figure S1.
Figure S2.
Figure S3.
Figure S4.
Figure S5.
A.
B.
Figure S6.
Figure S7.