(A) Silver-stained gel of glycerol-gradient fractions. Fifteen micrograms of recombinant DmORC were fractionated on a 4-ml glycerol gradient; 100-μl fractions were collected from the top of the gradient, and 10 μl of the peak containing fractions were separated on a 9% SDS/polyacrylamide gel and stained with silver. Fractions 16 to 19 were pooled and used for subsequent studies. (B) Silver-stained gel of recombinant purified wild-type and mutant DmORC proteins. Left to right: wild-type complex, ORC1A (K604A) mutant complex, ORC1B (DE684/685AA) mutant complex, ORC4A (K62A) mutant complex, ORC4B (EE147/148AA) mutant complex, ORC5A (K47A) mutant complex, and ORCΔ6 complex deficient for ORC6 subunit. In each lane, 100 ng of proteins were loaded.

ATP-dependent DNA binding of wild-type and mutant ORCs. Binding to a radiolabeled ori-β fragment (see Materials and Methods) was monitored in electrophoretic mobility shift assays. (A) ORC-wt was tested for DNA binding without ATP (lanes 2 and 9), with 0.5 mM ATP (lane 3), or with 0.5 mM ATPγS (lanes 4 and 10–13). Addition of monoclonal (lane 11) or of affinity-purified polyclonal (lane 12) antibodies against ORC2, but not a control antibody (lane 13), supershift the observed ATP-dependent protein–DNA complex. Controls: lanes 1 and 5, no protein; lanes 6–8, antibodies without addition of ORC. Arrows indicate the positions of unbound DNA and ORC–DNA complexes in the gel. (B) ORC-wt (lanes 1–10), ORC-1A (lanes 11–16), and ORC1B (lanes 17–22) were tested for DNA binding in the presence of increasing concentrations of ATP as indicated. (C) Wild-type ORC (lanes 2 and 3), ORC1A (lanes 4 and 5), ORC1B (lanes 6 and 7), ORC4A (lanes 8 and 9), ORC4B (lanes 10 and 11), or ORC5A (lanes 12 and 13) was incubated in the absence (lanes 2, 4, 6, 8, 10, and 12) or presence (lanes 3, 5, 7, 9, 11, and 13) of 50 μM ATP. (D) ORC composed only of subunits ORC1–5 (ORCΔ6) was tested for DNA binding in the absence (lane 4) or presence of 0.5 mM ATP (lane 5) or ATPγS (lane 6) and in the presence of ATPγS and either affinity-purified polyclonal antibodies against ORC2 (lane 7) or control rabbit IgG (lane 8).

ATPase activity of recombinant wild-type and mutant DmORC proteins. DNA inhibits the ATPase activity of ORC. ORC-wt (A), ORC5A (B, upper two lanes), ORCΔ6 (B, lower two lanes), ORC4A (C, upper two lines), ORC1A (C, lower two lines), ORC4B (D, upper two lines), and ORC1B (D, lower two lines) were incubated with α-[³²P]ATP in the absence (■) or presence (▴) of ori-β-DNA. At indicated time points, reactions were stopped, and the amount of ADP produced was determined by TLC and quantified by PhosphoImager analysis. The average of two independent experiments and ranges are shown.

(A) Chromatin binding of Drosophila wild-type and mutant ORC proteins. Demembranated Xenopus sperm DNA was incubated for 30 min in Drosophila extract depleted of the membranes. Where indicated, extracts were immunodepleted for ORC by using antibodies raised against ORC2 and ORC6. Add back experiments were performed by addition of 50 or 150 ng of recombinant wild-type or mutant ORC. Lane C is endogenous ORC binding to Xenopus chromatin. Chromatin-associated proteins were extracted with SDS/PAGE sample buffer, separated on a polyacrylamide gel, and subjected to a Western blotting reaction using antibody against ORC2. (B) DNA replication in Drosophila extracts. Xenopus sperm DNA was incubated for 1 h in Drosophila extract (with membranes) at a concentration of 2–5 ng/μl in a presence of [³²P]dCTP. Where indicated, extracts were depleted for ORC by using antibodies raised against ORC2 and ORC6. Add back experiment was performed by addition to depleted extracts of 50 or 150 ng of recombinant ORC proteins. (C) Density substitution analysis of replicated DNA. Demembranated Xenopus sperm DNA was incubated for 1 h in Drosophila egg extract at a concentration of 10 ng/μl in a presence of BrdUTP and [³²P]dCTP. DNA was extracted and subjected to centrifugation through gradient of CsCl. Xenopus sperm DNA in ORC-depleted extracts (blue) and after addition of 150 ng of recombinant wild-type (black) or recombinant mutant ORC (red, Walker A mutants or ORCΔ6 complex; green, Walker B mutations) are presented on density profiles. LL indicates the position of light–light DNA; HL shows the position of heavy–light DNA.

(A) Crude Drosophila egg extract separated on S300 Sephacryl column. Proteins from indicated fractions were separated on SDS gel and subjected to Western blot analysis using antibody raised against ORC2, ORC4, and ORC6. Markers used during fractionations were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and ovalbumin (43 kDa). (B) Localization of Drosophila ORC subunits in L2 cells. GFP-tagged Drosophila ORC1, ORC2, and ORC6 gene constructs were transiently transfected into Drosophila L2 cells. After 48 h, cells were fixed by using 1–3% paraformaldehyde and subsequently subjected to immunofluorescent microscopy (Carl Zeiss Axioplan, 100× magnification). (Scale Bar, 10 μm.) In vivo localization of Drosophila ORC subunits in early embryos before (C) and after (D) cellularization. Confocal microscopy was performed by using a Carl Zeiss LSM 510 microscope (40× and 100× magnification). Immunostaining of the Drosophila embryos was performed by using affinity-purified antibody raised against Drosophila ORC2 and ORC6 proteins. Arrows indicate the same cells within the Drosophila embryo shown here.