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Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

Kawakami H, Ohashi E, Tsurimoto T, Katayama T - Front Microbiol (2016)

Bottom Line: All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex.Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells.The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University Fukuoka, Japan.

ABSTRACT
Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

No MeSH data available.


Related in: MedlinePlus

In vitro activities of mutant ORC proteins. (A) The ARS-binding activity of Orc1–5 was examined by an electrophoretic mobility shift assay using Cy5-labeled wild-type (WT) or mutant (A− B2− B3−) ARS1 DNA. (B–D) Repression of ORC ATPase activity by ARS1 DNA in an EOS-dependent manner. WT or mutant (–ACS) ARS1 DNA was incubated with WT ORC (B), ORC containing Orc1 K362A (C), or ORC containing Orc1 R367A (D), yielding ATPase rates in the absence of DNA of 0.33 (B), 0.28 (C), and 0.25 (D) pmol/min/pmol ORC, respectively.
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Figure 7: In vitro activities of mutant ORC proteins. (A) The ARS-binding activity of Orc1–5 was examined by an electrophoretic mobility shift assay using Cy5-labeled wild-type (WT) or mutant (A− B2− B3−) ARS1 DNA. (B–D) Repression of ORC ATPase activity by ARS1 DNA in an EOS-dependent manner. WT or mutant (–ACS) ARS1 DNA was incubated with WT ORC (B), ORC containing Orc1 K362A (C), or ORC containing Orc1 R367A (D), yielding ATPase rates in the absence of DNA of 0.33 (B), 0.28 (C), and 0.25 (D) pmol/min/pmol ORC, respectively.

Mentions: To assess if the purified wild-type and mutant ORCs can be used for downstream applications such as biochemical analyzes, we first performed an electrophoretic mobility shift assay using Orc1–5 and wild-type and mutant ARS1 DNA. ORC and Orc1–5 bind to ARS at the nanomolar level in S. cerevisiae (Speck et al., 2005; Chen et al., 2008). Indeed, an Orc1–5-dependent band shift was seen at concentrations ≤2 nM with wild-type ARS1, whereas such shifts were not observed with mutant ARS1 (Figure 7A).


Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

Kawakami H, Ohashi E, Tsurimoto T, Katayama T - Front Microbiol (2016)

In vitro activities of mutant ORC proteins. (A) The ARS-binding activity of Orc1–5 was examined by an electrophoretic mobility shift assay using Cy5-labeled wild-type (WT) or mutant (A− B2− B3−) ARS1 DNA. (B–D) Repression of ORC ATPase activity by ARS1 DNA in an EOS-dependent manner. WT or mutant (–ACS) ARS1 DNA was incubated with WT ORC (B), ORC containing Orc1 K362A (C), or ORC containing Orc1 R367A (D), yielding ATPase rates in the absence of DNA of 0.33 (B), 0.28 (C), and 0.25 (D) pmol/min/pmol ORC, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834435&req=5

Figure 7: In vitro activities of mutant ORC proteins. (A) The ARS-binding activity of Orc1–5 was examined by an electrophoretic mobility shift assay using Cy5-labeled wild-type (WT) or mutant (A− B2− B3−) ARS1 DNA. (B–D) Repression of ORC ATPase activity by ARS1 DNA in an EOS-dependent manner. WT or mutant (–ACS) ARS1 DNA was incubated with WT ORC (B), ORC containing Orc1 K362A (C), or ORC containing Orc1 R367A (D), yielding ATPase rates in the absence of DNA of 0.33 (B), 0.28 (C), and 0.25 (D) pmol/min/pmol ORC, respectively.
Mentions: To assess if the purified wild-type and mutant ORCs can be used for downstream applications such as biochemical analyzes, we first performed an electrophoretic mobility shift assay using Orc1–5 and wild-type and mutant ARS1 DNA. ORC and Orc1–5 bind to ARS at the nanomolar level in S. cerevisiae (Speck et al., 2005; Chen et al., 2008). Indeed, an Orc1–5-dependent band shift was seen at concentrations ≤2 nM with wild-type ARS1, whereas such shifts were not observed with mutant ARS1 (Figure 7A).

Bottom Line: All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex.Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells.The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University Fukuoka, Japan.

ABSTRACT
Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

No MeSH data available.


Related in: MedlinePlus