Abstract
The Saccharomyces cerevisiae origin recognition complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multiprotein complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a complex with the five other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications.
Highlights
The replication of eukaryotic DNA is dependent upon the timely and accurate formation of a series of multiprotein assemblies
Biotinylated origin recognition complex (ORC) Complexes—To generate ORC complexes with a single subunit biotinylated at its C or N terminus, the last 88 residues of the acetyl-CoA carboxylase, biotin carboxyl carrier protein (BCCP) subunit (SMEAPAAAEISGHIVRSPMVGTFYRTPSPDAKAFIEVGQKVNVGDTLCIVEAMKMMNQIEADKSGTVKAILVESGQPVEFDEPLVVIE) [17], plus a two-amino acid linker (GG) were placed on either the C or N terminus of each ORC subunit, and that ORC subunit along with the remaining five nontagged subunits were expressed in insect cells [18] and purified as described [9]
A nanoscale biopointer consisting of a short stiff DNA with streptavidin at one end that could be seen in the EM was used to map the location of the C and N termini of each subunit in ORC
Summary
Biotinylated ORC Complexes—To generate ORC complexes with a single subunit biotinylated at its C or N terminus, the last 88 residues of the acetyl-CoA carboxylase, BCCP subunit (SMEAPAAAEISGHIVRSPMVGTFYRTPSPDAKAFIEVGQKVNVGDTLCIVEAMKMMNQIEADKSGTVKAILVESGQPVEFDEPLVVIE) [17], plus a two-amino acid linker (GG) were placed on either the C or N terminus of each ORC subunit, and that ORC subunit along with the remaining five nontagged subunits were expressed in insect cells [18] and purified as described [9]. To form the biopointers, 1 g of the DNA in TE was incubated with a 4-fold molar excess of streptavidin tetramers (Invitrogen) for 20 min at room temperature. Formation of ORC-Biopointer and ORC-Biopointer-DNA Complexes—Biopointers were incubated with purified ORC particles in 50 l of binding buffer (20 mM HEPES-KOH, pH 7.6, 2 mM EDTA, 5 mM magnesium acetate, 0.15 M KCl) containing 100 M ATP, 530 fmol of ORC (based on a molecular weight of 414,600 Da), and 1360 fmol of biopointers (based on a molecular mass of 184,100 Da). Following an overnight incubation at 4 °C, the samples were fixed with glutaraldehyde (0.6% final concentration) for 5 min at room temperature and filtered through a Chroma-SpinϩTE-100 column. Glutaraldehyde was added to a final concentration of 0.3% for 3 min at room temperature, followed by filtration through a Chroma-SpinϩTE-100 column. Images for publication were captured on sheet film, scanned with a Nikon LS4500 film scanner, the contrast optimized, and panels arranged using Adobe Photoshop software
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