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. 2011 May;39(9):3621-31.
doi: 10.1093/nar/gkq1308. Epub 2011 Jan 11.

Molecular Determinants of Origin Discrimination by Orc1 Initiators in Archaea

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Free PMC article

Molecular Determinants of Origin Discrimination by Orc1 Initiators in Archaea

Erin C Dueber et al. Nucleic Acids Res. .
Free PMC article

Abstract

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA(+)) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA(+) domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA(+) domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.

Figures

Figure 1.
Figure 1.
Initiator-origin interactions. (A) Schematic of S. solfataricus oriC2. Orc1-1-bound mORB sites are shown in purple, Orc1-3-bound C3 sites are shown in teal, the Orc1-2-bound C2 sites are shown in gray. The AT-rich DUE element is depicted as a gray box while the overlapping mORBa/C3b sites are highlighted by a dashed-red box. (B) The four residues observed to make base-specific contacts in the Orc1-1/Orc1-3/origin DNA structure (PDB 2QBY and 17) are highlighted in orange on a cartoon of the complex. Residues that make non-specific interactions but which are proposed to be important to origin recognition, are colored yellow. Protein, purple and teal; DNA, gray; ADP, black sticks; magnesium ions, magenta spheres. Mutations of arginine and asparagine residues in the WHDs to alanines are designed to weaken DNA binding by disrupting hydrogen bond interactions, whereas mutations of glycine-leucine/isoleucine sequences within the AAA+ ISMs (to leucine–aspartate or aspartate–aspartate pairs) are intended to disturb DNA interactions through steric hinderance and charge repulsion with the DNA phosphate backbone.
Figure 2.
Figure 2.
Orc1 binding to origin DNA sequences. Fluorescence anisotropy values of short, fluorescein-labeled origin DNA sequences are plotted as a function of increasing (A) Orc1-1 and (B) Orc1-3 variant concentration. As fluorescence anisotropy increases with the decrease in tumbling rate of the labeled species, DNA bound to Orc1 protein will have higher anisotropy values than free DNA. Only a subset of Orc1 variants is shown for clarity. Data shown are averaged from triplicate experiments and fit to the single-site binding equation to determine affinity (Table 1), with the exception of WT Orc1-3 and G126L/I127D, which are fit to a two-site binding equation due to a weak second binding event (possibly the result of protein-protein interactions, not shown). Mutations within the WHD and AAA+ ISM have varying effects on the affinity of Orc1 protein for origin DNA.
Figure 3.
Figure 3.
Orc1 specificity for origin DNA sequences. Representative binding curves of (A) Orc1-1 and (B) Orc1-3 ISM mutants demonstrate tighter binding to origin sequences than to a randomized DNA sequence. As illustrated in the (C) summary plot of Kd ratios (Kd,random/Kd,origin), most Orc1 mutants decrease the specificity of Orc1 proteins for their cognate origin DNA recognition sequences (larger ratios reflect greater specificity). The measured percentage change in specificity is indicated above each bar.
Figure 4.
Figure 4.
DNA distortion monitored by Cu-OP footprinting. Cu-OP footprints of the (A–B) top and (C–D) bottom strands of a 70 bp fragment of origin DNA illustrate areas of protection and hypersensitivity upon binding of either individual Orc1 variants (A and C) or Orc1 pairings (B and D). Maxam-Gilbert AG sequencing is shown in the far left lane of panels (A) and (C). The location of Orc1-1 (purple) and Orc1-3 (teal) binding sites are marked according to the initiator-DNA complex structure. The control lane corresponds to a mock footprinting experiment with no protein added.
Figure 5.
Figure 5.
Heat map of Orc1-induced Cu-OP modifications. Positions along the origin DNA sequence that show increased Cu-Op modification (DNA distortion) for each individual Orc1 variant and Orc1 pairing, as compared to DNA alone control, are colored red. Areas of decreased modification (DNA protection) are colored blue. The strength of the modification of the (A) top or (B) bottom strand is reflected in the intensity of the color plotted. A color key on the bottom indicates the correspondence between color and percentage-difference in band intensity. Gray squares indicate regions of the DNA sequence that were not resolved in the footprinting gel. Squares boxed by a thin black line indicate regions of extended protection arising from the mutation of ISM elements. Orc1-1 (purple) and Orc1-3 (teal) binding sites, as defined by the initiator-DNA complex structure, are highlighted on the origin sequence, with base-specific contacts shown boxed.
Figure 6.
Figure 6.
Comparison of archaeal initiator and origin properties with other domains of life. (A) Alignment of archaeal and yeast origin sequences. The S. solfataricus miniORB site is shown aligned with ORB sequences from A. pernix, Archaeoglobus fulgidus, P. furiosus and to the ORC binding site of S. cerevisiae ARS1 (encircled area demarcates the B1 element). Shaded areas within the alignment denote positions of sequence homology (purine or pyrimidine conserved), with positions contacted by Orc1 wing, HTH and AAA+ elements within origin-Orc1 co-crystal structures colored dark green, dark purple and dark red, respectively (17,18). Homologous sequences are highlighted in lighter shades of these colors. The S. solfataricus sequence ‘GGA’ is colored pink to reflect the apparent site of Orc1-1 AAA+ binding in the absence of Orc1-3 (17). The global DNA bend point induced by S. solfataricus Orc1-1 is highlighted (black arrow, top), as are two particularly strong, ORC-induced DnaseI hypersensitive sites in the yeast ARS1 sequence (black arrows, bottom, 37,38). (B) A continuum of origin binding mechanisms. Archaea and yeast appear to lie in between the highly specific recognition of conserved sequence repeats by the bacterial DnaA initiator at one extreme and the absence of distinct origin sequences for ORC binding in metazoans at the other.

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