Analysis of mutant origin recognition complex with reduced ATPase activity in vivo and in vitro.
In eukaryotes, ORC (origin recognition complex), a six-protein complex, is the most likely initiator of chromosomal DNA replication. ORC belongs to the AAA(+) (ATPases associated with a variety of cellular activities) family of proteins and has intrinsic ATPase activity derived from Orc1p, one of its subunits. To reveal the role of this ATPase activity in Saccharomyces cerevisiae (baker's yeast) ORC, we mutated the Orc1p sensor 1 and sensor 2 regions, which are important for ATPase activity in AAA(+) proteins. Plasmid-shuffling analysis revealed that Asn(600), Arg(694) and Arg(704) are essential for the function of Orc1p. In yeast cells, overexpression of Orc1R694Ep inhibited growth, caused inefficient loading of MCM (mini-chromosome maintenance complex of proteins) and slowed the progression of S phase. In vitro, purified ORC-1R [ORC with Orc1R694Ep (Orc1p Arg(694)-->Glu mutant)] has decreased ATPase activity in the presence or absence of origin DNA. However, other activities (ATP binding and origin DNA binding) were indistinguishable from those of wild-type ORC. The present study showed that Arg(694) of the Orc1p subunit is important for the ATPase activity of ORC and suggests that this ATPase activity is required for efficient MCM loading on to origin DNA and for progression of S phase.
Takehara
Masaya
M
Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
Makise
Masaki
M
Takenaka
Hitomi
H
Asano
Teita
T
Mizushima
Tohru
T
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D004261:DNA Replication / Q000235:genetics; D004271:DNA, Fungal / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D015151:Immunoblotting; D009154:Mutation; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; C497214:ORC1 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D001216:Asparagine; D001120:Arginine; D000251:Adenosine Triphosphatases