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. 2015 Oct 12;5:14929.
doi: 10.1038/srep14929.

Specific Binding of Eukaryotic ORC to DNA Replication Origins Depends on Highly Conserved Basic Residues

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Free PMC article

Specific Binding of Eukaryotic ORC to DNA Replication Origins Depends on Highly Conserved Basic Residues

Hironori Kawakami et al. Sci Rep. .
Free PMC article

Abstract

In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC.

Figures

Figure 1
Figure 1. Structural models of ScOrc1.
(a) Cartoons summarizing the ORC binding regions in typical ARSs and the structural model of the ORC-Cdc6-ARS1 complex. ACSs in ARS1, ARS306, and ARS609 are shown on top. Orc1 and Orc2/3/4/5 are shown in pink and white, respectively. Possible locations of the A and B1 elements are indicated. For clarity, Orc6 and the WH domains of Orc2/3/4/5 are omitted. (b) Side view of Orc1 complexed with DNA (as indicated by a dotted rectangle in panel (a) rotated 90° around the vertical axis from panel (a). Possible loci of a linker region that connects BAH and AAA+ domains are indicated as blue lines. (c) Domain structure and prediction of secondary structure of ScOrc1. Representative domains and motifs are indicated along with the predicted score of each residue. Walker A and B indicate the ATP-binding motifs. See text for BAH, AAA+ and WH.
Figure 2
Figure 2. In silico analyses of Orc1.
(a) Multiple alignment of a BLAST search with ScOrc1201–420 is shown on top using a colored rectangular pixel for each residue. A magnified alignment of the basic patch is shown below. The residues assigned to each color are indicated by the legend at left *, conserved residues. Full details of the alignment are shown in Supplementary Data S1. (b) Multiple alignment of the basic patch of eukaryotic Orc1 homologs, including representative model species. NCBI accession numbers are indicated on the right. Residues are colored as in panel (a). Histidine, though less basic than lysine or arginine, can play a crucial role for DNA binding at physiological pH. *, Lys-362 and Arg-367 residues in ScOrc1 and equivalents in homologs.
Figure 3
Figure 3. In vivo activities of orc1 mutants.
(a) Plasmid-shuffle assay. The tester strain YB838 (orc1::hisG pSPB16) was transformed with derivatives of pSPB15 bearing the indicated orc1 alleles and incubated at 30 °C. Serially diluted cells were spotted on the indicated selective media and further incubated at 30 °C. (b) Flow cytometry analysis. YHK26 (orc1-161 his3::pRS403), YHK27 (orc1-161 his3::ORC1-HS), YHK28 (orc1-161 his3::orc1 K362A-HS), and YHK29 (orc1-161 his3::orc1 R367A-HS) were grown at 23 °C to early log phase and then further incubated at 35 °C. Samples were taken at the indicated time points after the temperature shift. (c) ChAP assay. Cells of YHK26 (orc1-161 his3::pRS403), YHK33 (orc1-161 his3::ORC1-His12), YHK34 (orc1-161 his3::orc1 K362A-His12), and YHK35 (orc1-161 orc1 R367A-His12) were grown at 23 °C and further incubated at 35 °C. The amounts of indicated chromosomal loci crosslinked to wild-type or mutant Orc1-His12 were quantified and shown as relative enrichment compared with those using YHK26 (n = 3; mean ± SE).
Figure 4
Figure 4. In vitro activities of ScORC purified from a mammalian overexpression system.
(a) Gel-filtration analysis of affinity-purified ScOrc1-TEV-His co-overexpressed with ScOrc2/3/4/5/6 in 293T cells. Fractions were analyzed by 9% SDS-PAGE, followed by Coomassie staining. Gel positions of the ORC subunits are indicated. (b) Purified wild-type ORC, ORC-1K362A, and ORC-1R367A (1.2 pmol each) were analyzed by 9% SDS-PAGE, followed by Coomassie staining. (c) EMSA of Cy5-labeled DNA with wild-type ORC. DNA fragments contained wild-type (WT) or mutant (A B2 B3) ARS1. Protein concentrations are indicated. (d) EMSA of Cy5-labeled DNA, as in panel c, in the presence of wild-type ORC and Cdc6. (e) Affinities for ATP. Wild-type ORC or mutants (2.4 pmol each) were incubated with the indicated amount of [α-32P]ATP, and bound nucleotide was quantified by filter binding assay. (f) EMSA of Cy5-labeled ssDNA with wild-type ORC or mutants.
Figure 5
Figure 5. Affinities of ScORC for dsDNAs.
(a) EMSA of Cy5-labeled wild-type (WT) or mutant (A B2 B3) ARS1 DNA with wild-type or mutant ORC in the presence of competitor DNA. (b) Quantified results from panel (a) are shown. (c) EMSA of Cy5-labeled ARS306 or ARS609 DNA with wild-type or mutant ORC in the presence of competitor DNA. Wild-type (WT) or mutant (A B2 B3 or mut) ARS1 was also used as controls. (d) Quantified results from panel c are shown. (e,f) EMSA of Cy5-labeled DNA in the absence of competitor DNA. DNAs were the same as in panels (a,b).
Figure 6
Figure 6. Affinities of Orc1301–400 for dsDNAs.
(a) Wild-type and mutant GH-tagged Orc1301–400 (WT and R367A; 0.5 μg each) were analyzed by 12% SDS-PAGE, followed by Coomasie Brilliant Blue staining. (b) EMSA of Cy5-labeled mutant (A B2 B3) ARS1 DNA with wild-type or mutant GH-Orc1301–400 in the absence of competitor DNA. (c) EMSA of Cy5-labeled wild-type ARS1 DNA with ORC hexamer or GH-Orc1301–400 in the presence of competitor DNA. (d) EMSA of Cy5-labeled wild-type (WT) or mutant (A B2 B3) ARS1 DNA with wild-type or mutant GH-Orc1301–400 in the presence of competitor DNA.
Figure 7
Figure 7. Model for EOS-mediated origin recognition in eukaryotic ORC.
(a) A cartoon of ORC illustrating recognition of the A element via EOS and other domains in a mutually supportive manner. Orc1 and Orc2/3/4/5 are shown in pink and white, respectively. For clarity, Orc6 and the WH domains of Orc2/3/4/5 are omitted. (b) An atomic model predicts putative location of EOS. EOS (yellow) complexed with ACS DNA (orange) was computer-modeled and superimposed on the crystal structures of S. solfataricus Orc1-3 bound to DNA (PDB ID: 2QBY) and ScOrc1 BAH domain (green; PDB ID: 1M4Z). The AAA+ and WH domains and DNA in the crystal structure are shown in magenta, blue, and gray, respectively. See Discussion for details.

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