Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Dec;164(3):241-9.
doi: 10.1016/j.jsb.2008.08.006. Epub 2008 Sep 11.

Single Particle EM Studies of the Drosophila Melanogaster Origin Recognition Complex and Evidence for DNA Wrapping

Affiliations
Free PMC article

Single Particle EM Studies of the Drosophila Melanogaster Origin Recognition Complex and Evidence for DNA Wrapping

Megan G Clarey et al. J Struct Biol. .
Free PMC article

Abstract

Hyperphosphorylation of the Drosophila melanogaster origin recognition complex (DmORC) by cyclin dependent kinases (CDKs) allows nucleotide binding but inhibits the ATPase activity of Orc1, and ablates the ATP-dependent interaction of ORC with DNA. Here we present single particle electron microscopy (EM) studies of ORC bound to nucleotide in both the dephosphorylated and hyper-phosphorylated states. 3D image reconstructions show that nucleotide binding gives rise to an analogous conformation independent of phosphorylation state. At the intermediate resolution achieved in our studies, ATP promotes changes along the toroidal core of the complex with negligible differences contributed by phosphorylation. Thus, hyperphosphorylation of DmORC does not induce meso-scale rearrangement of the ORC structure. To better understand ORC's role in origin remodeling, we performed atomic force microscopy (AFM) studies that show the contour length of a 688bp linear DNA fragment shortens by the equivalent of approximately 130bp upon ORC binding. This data, coupled with previous studies that showed a linking number change in circular DNA upon ORC binding, suggests that ORC may wrap the DNA in a manner akin to DnaA. Based on existing data and our structures, we propose a subunit arrangement for the AAA+ and winged helix domains, and in addition, speculate on a path of the 133bp of DNA around the ORC complex.

Figures

Fig. 1
Fig. 1. Hyper-phosphorylated versus dephosphorylated Drosophila melanogaster ORC
(A) Silver stain of SDS–PAGE exhibiting the molecular weight shift in the Orc1 and Orc2 subunits in dephosphorylated versus hyper-phosphorylated ORC. Orc1 has seven phosphorylation sites while Orc2 has four phosphorylation sites. (B) Audioradiogram of Cyclin E/CDK2 hyperphosphorylation of recombinant DmORC showing 32P signal versus increasing amounts of Cyclin E/CDK2. (C) Representative two-dimensional class averages from single particle electron microscopy reference-free image classification of dephosphorylated apo-ORC, ATPγS bound dephosphorylated ORC and ATPγS bound hyper-phosphorylated ORC. All three data sets show preferential orientations around the elongated axis of ORC.
Fig. 2
Fig. 2. Three-dimensional comparison of ORC indicates ATP gives rise to analogous conformational changes in the core of ORC independent of phosphorylation state. All volumes were filtered to 30 Å and are displayed in four identical views rotated 90 ° counter-clockwise around their elongated axis
(A) Dephosphorylated ORC is rendered as a blue isosurface. The five lobes of density composing the core are labeled A–E. A thin density closes the central channel and fuses the shoulder to lobe A. The glove density at the top remains separate from the main body of the complex. (B) Dephosphorylated ORC bound to ATPγS is rendered as a red isosurface. An arrow points to the region where upon nucleotide-binding a discernable stalk density separates lobe D from E. The shoulder is again fused with lobe A and the glove is now connected to lobe E. The density labeled lobe E was previously identified as Orc5 by immuno-labeling. (C) Hyper-phosphorylated ORC bound to ATPγS is rendered as a green isosurface. The separation of lobes D and E as well as the fusing of the glove with lobe E is also observed, indicating that nucleotide binding promotes a very similar conformational state in the core of ORC regardless of the phosphorylation state of the complex.
Fig. 3
Fig. 3. ATP-binding leads to similar ORC conformations, independent of phosphorylation state
(A) Pairwise fourier shell correlation comparison of dephosphorylated ORC versus dephosphorylated ORC bound to ATPγS (red line), dephosphorylated ORC versus hyper-phosphorylated ORC bound to ATPγS (yellow line), and dephosphorylated ORC bound to ATPγS versus hyper-phosphorylated ORC bound to ATPγS (blue line). Apo-dephosphorylated ORC has a distinctly different conformation as compared to the ATP-bound volumes, as is indicated by the highest correlation between the two ATPγS volumes. The correlation between dephosphorylated ORC and nucleotide-bound hyperphopshorylated ORC is the weakest, suggesting additional small differences due to the phosphorylation state, in addition to those larger differences caused by nucleotide binding. (B) Difference maps showing the main regions of difference between the complexes. Only positive differences are shown for clarity. Left, the volume of the ATP-bound dephosphorylated ORC (grey mesh isosurface) was subtracted from that of the dephosphorylated ORC without added nucleotide (transparent grey isosurface) with the mass differences highlighted in orange. The main areas of observed differences are, from the top and moving clockwise: (1) on the top of the glove density, (2) in the glove-shoulder connection, (3) inside the central channel, (4) the movement of A towards the shoulder, (5) minor changes at the bottom of the complex in the connection of the toroidal AAA+ core and the collar, and (6) the separation of D and E that gives rise to the stalk. Right, the ATP-bound hyper-phosphorylated ORC volume (grey mesh isosurface) was subtracted from the dephosphorylated ORC volume (transparent grey isosurface) with the mass differences highlighted in green. The similarity in the position of the mass differences corresponding to positions 2, 3, 5 and 6 exemplifies the analogous effect of nucleotide addition independent of hyperphosphorylation in ORC. While positions 1* and 4* do not have large mass differences, they have conformations distinct from the apo-dephosphorylated volume that are more closely related to the nucleotide-bound dephosphorylated volume.
Fig. 4
Fig. 4. ORC wraps ~130 bp of linear DNA. Atomic force microscopy images of linear 688 bp DNA containing the ACE-3 Chorion gene sequence plus and minus DmORC on APS-mica
(A) representative 688 bp linear DNA fragments bound to a DmORC (top four panels) and alone (bottom two panels). Asterisk (*) indicates a DNA fragment with two ORCs bound. Scale bar correlating to the theoretical length of 688 bp of DNA or 234 nm. (B) Graph of the average length of a linear 688 bp DNA fragment with (black columns) and without (grey columns) ATPγS DmORC bound. The measured lengths of linear 688 bp DNA were binned into 10 nm groups. The average measured length of an unbound 688 bp DNA fragment was ~ 258 nm while the average length of the 688 bp DNA fragment with DmORC bound was 213 nm. The average difference in length of DNA with DmORC bound is 45 nm or ~133 bp.
Fig. 5
Fig. 5. Speculative model of ORC subunit architecture and the path of 133 bp of DNA
(A) A potential arrangement of the subunits would have the five AAA+ domains (illustrated as green spheres) of Orc1–5 composing the core of the complex, with Orc1 situated at the base directly contacting Orc4, Orc2 and Orc3 in the central region where the stalk density shifts upon nucleotide binding, followed by Orc5. The position of Orc2 and Orc3 are interchangeable and are labeled “2/3” to indicated this in the figure. The winged helix domains (illustrated as yellow spheres) of Orc1, 4 and 5 would form the collar. This position would allow for the AAA+ domain of Cdc6 to dock at the AAA+ interface of Orc1. The mass of the other domains of Orc1–5 and Orc6 would compose the remainder of the density. (B) A model of dephosphorylated ORC bound to ATPγS (rendered as a grey isosurface) with 133 bp of a linear DNA wrapped around the complex starting at the wing-helix domains in the collar and following a right-handed path along the AAA+ backbone to loop back through the channel.

Similar articles

See all similar articles

Cited by 20 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback