Mapping subunit location on the Saccharomyces cerevisiae origin recognition complex free and bound to DNA using a novel nanoscale biopointer.
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.
Chastain
Paul D
PD
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.
Bowers
Jayson L
JL
Lee
Daniel G
DG
Bell
Stephen P
SP
Griffith
Jack D
JD
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008957:Models, Genetic; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013997:Time Factors
D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D001710:Biotin; D000255:Adenosine Triphosphate; D004247:DNA