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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

Cartoons summarizing ORC-ARS binding. (A) The ORC-binding regions of ARS. The A and B1 elements and the ARS consensus sequence are indicated. (B) Direct recognition of the A element by EOS with a low affinity. Orc1 and Orc2/3/4/5 are shown in pink and white, respectively. Orc6 and the WH domains of Orc2/3/4/5 are omitted for clarity. (C) Recognition of the A element via EOS and other domains in a mutually supportive manner with a higher affinity. The initial interaction of EOS with the A element leads to additional interactions with other domains (possibly AAA+ and WH domains), resulting in high-affinity binding. The ORC subunits are shown as in panel (B).
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Figure 1: Cartoons summarizing ORC-ARS binding. (A) The ORC-binding regions of ARS. The A and B1 elements and the ARS consensus sequence are indicated. (B) Direct recognition of the A element by EOS with a low affinity. Orc1 and Orc2/3/4/5 are shown in pink and white, respectively. Orc6 and the WH domains of Orc2/3/4/5 are omitted for clarity. (C) Recognition of the A element via EOS and other domains in a mutually supportive manner with a higher affinity. The initial interaction of EOS with the A element leads to additional interactions with other domains (possibly AAA+ and WH domains), resulting in high-affinity binding. The ORC subunits are shown as in panel (B).

Mentions: The origin recognition complex (ORC), consisting of Orc1/2/3/4/5/6, is one such protein complex (Duncker et al., 2009; Kawakami and Katayama, 2010; Li and Stillman, 2012). ORC binds to eukaryotic chromosomal replication origins in an ATP-dependent manner to recruit Cdc6, Cdt1, and the MCM2-7 helicase core onto double-stranded DNA (Boos et al., 2012; Bell and Kaguni, 2013; Yardimci and Walter, 2014; Tognetti et al., 2015). In the budding yeast Saccharomyces cerevisiae, replication origins are called autonomously replicating sequences (ARSs). ARSs bear two major functional elements, namely, the essential A element containing the ARS consensus sequence and the stimulatory B elements (Figure 1). The A and B1 elements are essential for ORC binding. All ORC subunits except for Orc6 are highly conserved among eukaryotes and belong to the AAA+ (ATPases associated with a variety of cellular activities) superfamily, although only Orc1 and Orc5 bind to ATP. ORC ATPase activity is repressed by ARS DNA in vitro and thought to ensure timely recruitment of the MCM2-7 helicase. Orc1/2/3/4/5 also bear one or two winged-helix DNA-binding motifs at the C-terminus. Orc1 bears an extension at the N-terminus called the BAH (bromo-adjacent homology) domain that binds to transcription-related proteins. The linker region between BAH and AAA+ bears a highly conserved, basic residue-rich motif called the eukaryotic origin sensor (EOS) that solely and directly scans the essential element in ARS with a low affinity to achieve high-affinity binding of the ORC hexamer to ARS (Figure 1; Kawakami et al., 2015). Elimination of one subunit (except for Orc6) from the ORC hexamer abolishes high-affinity binding of ORC to ARS (Lee and Bell, 1997), suggesting that purification of the entire ORC complex rather than individual subunits is important to analyze the biological functions of ORC.


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

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

Cartoons summarizing ORC-ARS binding. (A) The ORC-binding regions of ARS. The A and B1 elements and the ARS consensus sequence are indicated. (B) Direct recognition of the A element by EOS with a low affinity. Orc1 and Orc2/3/4/5 are shown in pink and white, respectively. Orc6 and the WH domains of Orc2/3/4/5 are omitted for clarity. (C) Recognition of the A element via EOS and other domains in a mutually supportive manner with a higher affinity. The initial interaction of EOS with the A element leads to additional interactions with other domains (possibly AAA+ and WH domains), resulting in high-affinity binding. The ORC subunits are shown as in panel (B).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Cartoons summarizing ORC-ARS binding. (A) The ORC-binding regions of ARS. The A and B1 elements and the ARS consensus sequence are indicated. (B) Direct recognition of the A element by EOS with a low affinity. Orc1 and Orc2/3/4/5 are shown in pink and white, respectively. Orc6 and the WH domains of Orc2/3/4/5 are omitted for clarity. (C) Recognition of the A element via EOS and other domains in a mutually supportive manner with a higher affinity. The initial interaction of EOS with the A element leads to additional interactions with other domains (possibly AAA+ and WH domains), resulting in high-affinity binding. The ORC subunits are shown as in panel (B).
Mentions: The origin recognition complex (ORC), consisting of Orc1/2/3/4/5/6, is one such protein complex (Duncker et al., 2009; Kawakami and Katayama, 2010; Li and Stillman, 2012). ORC binds to eukaryotic chromosomal replication origins in an ATP-dependent manner to recruit Cdc6, Cdt1, and the MCM2-7 helicase core onto double-stranded DNA (Boos et al., 2012; Bell and Kaguni, 2013; Yardimci and Walter, 2014; Tognetti et al., 2015). In the budding yeast Saccharomyces cerevisiae, replication origins are called autonomously replicating sequences (ARSs). ARSs bear two major functional elements, namely, the essential A element containing the ARS consensus sequence and the stimulatory B elements (Figure 1). The A and B1 elements are essential for ORC binding. All ORC subunits except for Orc6 are highly conserved among eukaryotes and belong to the AAA+ (ATPases associated with a variety of cellular activities) superfamily, although only Orc1 and Orc5 bind to ATP. ORC ATPase activity is repressed by ARS DNA in vitro and thought to ensure timely recruitment of the MCM2-7 helicase. Orc1/2/3/4/5 also bear one or two winged-helix DNA-binding motifs at the C-terminus. Orc1 bears an extension at the N-terminus called the BAH (bromo-adjacent homology) domain that binds to transcription-related proteins. The linker region between BAH and AAA+ bears a highly conserved, basic residue-rich motif called the eukaryotic origin sensor (EOS) that solely and directly scans the essential element in ARS with a low affinity to achieve high-affinity binding of the ORC hexamer to ARS (Figure 1; Kawakami et al., 2015). Elimination of one subunit (except for Orc6) from the ORC hexamer abolishes high-affinity binding of ORC to ARS (Lee and Bell, 1997), suggesting that purification of the entire ORC complex rather than individual subunits is important to analyze the biological functions of ORC.

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