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Fig. 1. Dominant lethal when overexpressed mutants within ORC1. Strains containing wild-type ORC1 (upper left), a mutant form of ORC1 that cannot bind ATP (orc1-K485T, upper right), orc1-d1 (D569Y, lower right), or orc1-d2 (D569F, lower left) under the control of the GAL1–10 promoter were grown on a plate containing galactose (Lower), which induces overexpression, or glucose (Upper), which does not induce overexpression. All strains also overexpressed wild-type ORC2–ORC6 genes when grown on galactose. ORC1 that cause lethality when overexpressed. Our strategy was based on two observations concerning other ATPases. First, studies of other ATPases indicate that mutations that cause lethality when overexpressed frequently are able to bind but not hydrolyze ATP. Second, mutations in the Walker B motif of other ATPases frequently inhibit the ATPase activity but not the ATP binding activity of these proteins. Because Orc1p is stably associated with the other five ORC subunits and has no apparent activity on its own (ref. 23 and data not shown), the screen was performed in a strain that simultaneously overexpressed the mutant Orc1p and wild-type Orc2p-Orc6p after induction with galactose. We first tested whether overexpression of wild-type ORC caused lethality, because it was possible that ORC overexpression could titrate critical replication factors away from the origin. There was no growth defect caused by wild-type ORC overexpression (Fig. 1). We also tested whether a previously identified mutant in Orc1p that prevents ATP binding (and hydrolysis) was dominant lethal (14). We found that this mutant, orc1-K485T, also did not exhibit a dominant lethal phenotype when overexpressed (Fig. 1). Having found that neither wild-type ORC nor ORC containing an Orc1p ATP-binding mutant showed a dominant negative phenotype, we initiated a screen to identify mutations in the Orc1p Walker B motif that exhibited such a phenotype. Four conserved amino acid residues within the Walker B motif (DELD) were randomly mutagenized (see Materials and Methods). We individually tested 80 random mutants targeted to each of the four amino acids (320 total) and found 18 that resulted in lethality or decreased viability when overexpressed. The seven mutants that caused a complete loss of viability were sequenced. Of these, four corresponded to a change of the second aspartate to tyrosine (D569Y, named Orc1-d1) and three corresponded to a change of the second aspartate to phenylalanine (D569F, named Orc1-d2). Because these alleles were obtained multiple times with different codons encoding the tyrosine or the phenylalanine, we believe that the library of orc1 mutants was saturated. Interestingly, no dominant mutants were identified in the first two amino acid positions, which are typically the most well conserved among ATP binding proteins. The lack of mutants in this region may be due to inhibition of ATP binding, because previous studies of ORC complexes mutated in these amino acids resulted in a loss of ATP binding as well as ATP hydrolysis (data not shown). Consistent with the hypothesis that the lethal phenotype was due to the overexpression of an intact mutant ORC complex, the dominant lethal phenotype of Orc1-d1 and Orc1-d2 required co-overexpression of ORC2–6 (data not shown). ORC1-d1 Has a Hydrolysis Defect in Vitro. To understand the molecular defect associated with these dominant lethal alleles of Orc1p, we expressed and purified the mutant orc1-d1 encoded subunit complexed with the remaining five ORC subunits and characterized this mutant ORC complex (for simplicity hereafter we refer to this complex as ORC-d1 and the complex containing Orc1-d2p as ORC-d2) in vitro. The ORC complex used for these in vitro studies contained a second mutation in the Walker A motif of Orc5p to focus the analysis on ATP binding to Orc1p. Orc5p binds ATP in a DNA-independent manner, and does not hydrolyze ATP at detectable levels (14). We are confident that this change in the complex did not affect our studies of Orc1p function because Orc5p mutants that cannot bind ATP support viability, indicating that ATP binding to Orc5p is not required for any essential ORC function. ATP hydrolysis activity was measured for an ORC complex containing wild-type Orc1p, a complex with a point mutation in the Orc1p Walker A motif that eliminates ATP binding (orc1-K485T, ORC-1A), and the ORC-d1 complex (Fig. 2A). As shown previously, ATP hydrolysis by wild-type Orc1p is inhibited by autonomously replicating sequence-1 (ARS1) origin DNA. The ORC-1A complex is defective for ATP hydrolysis both in the presence and absence of ARS1 DNA. The mutant ORC-d1 complex hydrolyzed ATP at a 16-fold reduced rate in the absence of ARS1 DNA. In contrast, in the presence of ARS1 DNA, the ORC1-d1 mutant could hydrolyze ATP at a rate slightly higher than wild-type ORC. The loss of hydrolysis activity by the ORC-d1 complex could be due to a defect in binding ATP or in the rate of catalysis. To clarify this issue, we measured the ATPase activity of the wild-type and ORC-d1 complexes at various concentrations of ATP to determine KM and kcat. Our findings are shown as an Eadie-Hofstee plot (Fig. 2B). The complex containing wild-type Orc1p had a KM of 5 µM and a kcat of 0.16 min−1. In contrast, the ORC-d1 complex had a KM of 50 µM and a kcat of 0.01 min−1. These results indicate that the mutant has defects in both the binding component and the catalysis component of the reaction. However, the concentration of ATP in the cell is between 1.5 mM and 4.0 mM (24, 25)—at least 30-fold higher than the KM of this mutant. At this concentration of ATP, the ORC-d1 complex is expected to be >96% saturated with ATP (vs. >99% for wild-type) but have a 16-fold hydrolysis defect. Thus, the primary defect of ORC-d1 at physiological concentrations of ATP is a reduced rate of ATP hydrolysis, not ATP binding. To detect ATP binding to Orc1p, we assayed the ability of the wild-type and mutant ORC complexes to bind origin DNA by using a DNase I protection assay of ARS1 (origin DNA binding is ATP dependent, Fig. 2C). At concentrations of 100 µM and 1.0 mM, the wild-type and ORC-d1 complexes can efficiently bind DNA, yet no origin binding is seen in the absence of ATP. As shown previously, The ORC-1A mutant is defective in origin binding. Thus, ORC-d1 and wild-type ORC have approximately equivalent DNA and ATP binding activities at physiological ATP levels. When not bound to DNA, our analysis of ATP
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