Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping.
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.
Clarey
Megan G
MG
Molecular & Cell Biology Department, University of California at Berkeley, Berkeley, CA 94720, USA.
Botchan
Michael
M
Nogales
Eva
E
D000818:Animals; D002875:Chromosomes / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008968:Molecular Conformation; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism; D051738:Origin Recognition Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation
D009711:Nucleotides; C497223:ORC1 protein, human; D051738:Origin Recognition Complex; D004247:DNA; D018844:Cyclin-Dependent Kinases