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. 2007 Apr;27(8):3143-53.
doi: 10.1128/MCB.02382-06. Epub 2007 Feb 5.

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

Maxim Balasov  1 , Richard P H Huijbregts, Igor Chesnokov
Affiliations

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

Maxim Balasov et al. Mol Cell Biol. 2007 Apr.

Erratum in

  • Mol Cell Biol. 2007 Jun;27(11):4206

Abstract

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.

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Figures

FIG. 1.
FIG. 1.
DNA-binding ability of Drosophila Orc6 protein. Binding to radiolabeled origin and non-origin DNA fragments was monitored in EMSAs. (A) Binding of Orc6 protein to ori-β (lanes 1 to 8), ACE3 (lanes 9 to 12), ori-β-R (lanes 13 to 16), and Orc6 cDNA (lanes 17 to 19) DNA fragments. Orc6-wt (lanes 2 to 5, 9 to 11, 13 to 15, and 18 to 19) or the Orc6-200 deletion mutant (lanes 7 and 8) protein (50 ng each) was incubated with origin (ACE3 and ori-β) and non-origin (ori-β-R and Orc6 cDNA) DNA fragments in the presence of increasing amounts of competitor poly(dI-dC) DNA. The amount of competitor was 100 ng, 200 ng, 500 ng, and 1,000 ng (lanes 2, 3, 4, and 5, respectively) for the ori-β probe; 100 ng, 200 ng, and 500 ng (lanes 9, 10, and 11, respectively) for ACE3 probe; 100 ng, 200 ng, and 500 ng (lanes 13, 14, and 15, respectively) for ori-β-R probe; and 100 ng and 200 ng (lanes 18 and 19, respectively) for Orc6 cDNA probe. A total of 200 ng of competitor DNA was used in lanes 7 and 8. Addition of affinity-purified polyclonal antibodies against Orc6 supershifted the Orc6-200-DNA complex (lane 8). Controls: lanes 1, 6, 12, 16, and 17 (no protein). (B) Binding preferences of Orc6-wt protein. Orc6-wt (100 ng) was incubated with the ori-β fragment in the presence of increasing amounts of various competitor DNAs (100 ng, 500 ng, and 1,000 ng). IC, poly(dI-dC) (lanes 2 to 4); I/C, poly(dI) · poly(dC) (lanes 5 to 7); AT, poly(dA-dT) (lanes 8 to 10); A/T, poly(dA) · poly(dT) (lanes 11 to 13); GC, poly(dG-dC) (lanes 14 to 16); G/C, poly(dG) · poly(dC) (lanes 17 to 19). Lane 1, no protein.
FIG. 2.
FIG. 2.
Molecular modeling of the Orc6 structure and strategy for selecting point mutations. (A) Molecular model of the Orc6 protein in a complex with DNA. The structure is based on a predicted structural homology between Orc6 and TFIIB. The Swiss PDB program (21) (www.expasy.org/spdbv/) and PyMol program (Delano Scientific) (www.pymol.org) were used to dock Orc6 to the DNA scaffold. A putative helix-turn-helix motif of Orc6 that might be important for interaction with DNA is highlighted. (B) Amino acids serine 72 and lysine 76 are shown within a putative helix-turn-helix motif in the Orc6-DNA model. (C) Amino acid alignment of mouse, human, and Drosophila Orc6 proteins shows a conservative block between amino acids 69 and 79 (numbering is according to the Drosophila Orc6 sequence). Serine 72 and lysine 76 residues (indicated by stars) have been mutated to alanines.
FIG. 3.
FIG. 3.
(A) Silver-stained gel of purified wt and mutant Orc6 proteins. In each lane, 300 ng of protein was loaded. (B) DNA binding ability of wt and mutant Orc6 proteins. Proteins were incubated with ori-β fragment in the presence of increasing amounts of poly(dI-dC) competitor DNA (100 ng, 200 ng, 500 ng, and 1000 ng). A total of 100 ng of protein was used per reaction.
FIG. 4.
FIG. 4.
GFP fused Orc6-wt (A), Orc6-200 (B), Orc6-S72A (C), and Orc6-K76A (D) mutants expressed in salivary glands. We induced the expression of various GFP-Orc6 proteins in salivary gland of third-instar larvae to test chromosome binding of Orc6 mutants. Flies bearing GFP-fused Orc6 genes were crossed to P{Sgs3-GAL.PD}TP1 flies, and progeny of third-instar larvae were analyzed for GFP expression under a UV dissecting microscope. The Sgs-3 promoter drives GAL4 expression in salivary glands of third-instar larvae at high levels, which makes picking up live larvae for imaging easy. Salivary glands expressing GFP-fused Orc6 were analyzed as described in Materials and Methods.
FIG. 5.
FIG. 5.
Sites of active BrdU incorporation (marker for DNA replication) colocalize with the Orc6 protein in salivary gland polytene chromosomes. Immunostaining data are presented. Salivary glands were dissected in PBS, stained with antibody raised against Drosophila Orc6 protein (A) and anti-BrdU antibody (B) and counterstained with DAPI. A merged image is shown in panel C. White arrowheads indicate the examples of the colocalization. GFP-Orc6, overexpressed in Drosophila salivary glands using the GAL-4/UAS binary system, binds with chromosomes and prefers interband regions. DAPI staining is shown (D) as well as immunostaining with antibody raised against GFP protein (E). A merged image is shown in panel F. Bars and arrows indicate examples of heavily stained (DAPI) bands in regions 21C, 21D, 21E, 22A, 23A, 25A, and 31A (A) which do not contain Orc6 (E). The merged image confirms that the strong Orc6 signal comes from interband regions (F).
FIG. 6.
FIG. 6.
Drosophila Orc6 protein is important for ORC-dependent DNA binding and DNA replication in vitro. (A) Silver-stained gel of recombinant purified wt and mutant Drosophila ORC proteins. In each lane 100 ng of protein was loaded. (B) DNA binding of ORC6-wt and ORC containing mutant Orc6 proteins. ORC amounts added (50 ng, 100 ng, and 150 ng) are shown above the lanes. (C) DNA binding of the ORC with the Orc6-K76A mutant [ORC(6-K76A)] and the Orc6-S72A mutant [ORC(6-S72A)]) was tested at higher concentrations (150 ng, 300 ng, and 450 ng) of the protein. (D) DNA replication in Drosophila extracts. Xenopus sperm DNA was incubated for 1 h in Drosophila extract (with membranes) at a concentration of 2 to 5 ng/μl in the presence of [32P]dCTP. Where indicated, extracts were depleted of ORC by using antibodies raised against Orc2 and Orc6. An add-back experiment was performed by the addition of 50, 100, or 150 ng of recombinant ORC proteins to depleted extracts. RE, nondepleted replication extract control (lane 1).
FIG. 7.
FIG. 7.
BrdU incorporation in L2 cells expressing GFP-Orc6-wt and mutants GFP-Orc6-K76A, GFP-Orc6-S72A, and GFP-Orc6-200. GFP-tagged Drosophila Orc6 gene constructs under the control of the metallothionein promoter were transiently transfected into Drosophila L2 cells. The metallothionein promoter was induced by 0.5 mM CuSO4, and cells were incubated with BrdU overnight at a final concentration of 10 μM. Cells were fixed by using 2% paraformaldehyde, stained with anti-BrdU antibody, and subsequently subjected to immunofluorescent microscopy (magnification, ×40; Carl Zeiss Axioplan).
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