Isolation of
            <i>ORC6</i>
            , a Component of the Yeast Origin Recognition Complex by a One-Hybrid System

Li, Joachim J.; Herskowitz, Ira

doi:10.1126/science.8266075

Cited by 398 publications

(254 citation statements)

References 31 publications

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“…Yeast transformants that had been grown for 2 or 3 days at 30ЊC were overlaid with either a nitrocellulose membrane filter (Schleicher & Schuell) or Whatman #50 filter paper and incubated overnight at 30ЊC. The yeast-impregnated filters were lifted from the plates and treated with X-Gal (5-bromo-4-chloro-3-indolyl ␤-D-galactoside) to assay colonies for ␤-galactosidase activity (15). Plasmid DNA from transformants that formed blue colonies on X-Gal-treated filters was recovered (16), propagated in E. coli DH5␣, and transformed back into the yeast reporter strains to confirm activity.…”

Section: Methodsmentioning

confidence: 99%

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

Stockinger

Gilmour

Thomashow

1997

Proc. Natl. Acad. Sci. U.S.A.

1,590

1,203

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Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat͞dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat͞DRE binding factor, CBF1 (C-repeat͞DRE Binding Factor 1). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat͞DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli. Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat͞DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat͞DRE DNA regulatory element and, thus, is likely to have a role in cold-and dehydrationregulated gene expression in Arabidopsis.

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Section: Methodsmentioning

confidence: 99%

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

Stockinger

Gilmour

Thomashow

1997

Proc. Natl. Acad. Sci. U.S.A.

1,590

1,203

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“…Homologues of ORC1 (45) 5, and 9), 60 (lanes 2, 6, and 10), 150 (lanes 3, 7 and 11), and 300 (lanes 4, 8, and 12) required for DNA replication in an in vitro system. A genetic interaction between ORC components and MCM proteins has been reported in S. cerevisiae (44,47). Therefore, MCM protein complexes may be involved in the initiation of DNA replication as a DNA unwinding enzyme by interacting with ORC.…”

Section: Fig 3 Protein Cross-linking Of MCM Proteins a The Pooledmentioning

confidence: 99%

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

Ishimi

1997

Journal of Biological Chemistry

506

508

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All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals. Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S., Omori, A., Sato K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). The present study revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex. Both ATPase and DNA helicase activities were co-purified with a 600-kDa protein complex that is consisted of equal amounts of MCM4, -6, and -7 proteins. An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody resulted in the severe reduction of the DNA helicase activity. Displacement of DNA fragments by the DNA helicase suggested that it migrated along single-stranded DNA in the 3 to 5 direction, and the DNA helicase activity was detected only in the presence of hydrolyzable ATP or dATP. These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme.There are at least six MCM 1 proteins that play an essential role in eukaryotic DNA replication as follows: MCM2, -3, -4 (Cdc21), -5, -6 (Mis5), and -7 (CDC47) proteins (see reviews in Refs. 1-3). Genetic evidence suggests that these proteins are required for the initiation of DNA replication in yeast (4, 5). The inactivation of human MCM2 protein with antibody consistently inhibits the initiation of DNA replication (6). Interaction between the MCM proteins has been reported in yeast (4, 7), and the protein complexes containing MCM proteins are detected in Drosophila (8), Xenopus (9), mouse (10, 11), and human cells (12)(13)(14). In human cells, MCM4, -6, and -7 proteins form a stable complex, and MCM2 is loosely associated with this complex (12,15,16). MCM3 and -5 proteins also form a stable complex (10, 13). All six of the MCM proteins contain DNA-dependent ATPase motifs in a central domain that is conserved from yeast to mammals, and these motifs are found in several enzymes that unwind the DNA duplex (17). These findings suggest that MCM protein complexes may be involved in the initiation of DNA replication as an enzyme that unwinds DNA in the origin region. However, DNA helicase activity of MCM proteins has not been reported.MCM proteins bind with chromatin before the initiation of DNA replication and are then released from the chromatin as DNA replication proceeds (18 -20). Our group found that MCM protein complexes containing MCM2, -4, -6, and -7 proteins bound tightly with histone H3 in vitro and were purified to near homogeneity by histone-Sepharose column chromatography (21). In the present study, it is shown that both ATPase and DNA helicase activities are associated with an MCM4, -6, and -7 protein complex. EXPERIMENTAL PROCEDURESPurification of MCM ...

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“…Protein-binding microarrays (PBM) 12,13 are well suited to detect high affinity binding sequences for a given protein, if available in high amounts. Yeast-one-hybrid (Y1H) 14,15 and bacterial one-hybrid (B1H) 16 approaches are typically used to determine the proteins that bind to a given DNA sequence and are thus complementary to ChIP and PBM. High-throughput versions have been proposed.…”

Section: Dna-protein Studiesmentioning

confidence: 99%

Protein–DNA force assay in a microfluidic format

2013

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Protein-DNA interaction forces are studied using a miniaturized and multiplexed molecular force assay on a microfluidic MITOMI chip and with a new confocal analysis method. As featured in:See Marcus Otten et al., Lab Chip, 2013, 13, 4198. Cite this: Lab Chip, 2013 Protein-DNA force assay in a microfluidic format3 The detailed study of protein-DNA interactions is a core effort to elucidate physiological processes, including gene regulation, DNA repair and the immune response. The molecular force assay (MFA) is an established method to study DNA-binding proteins. In particular, high-affinity binder dissociation is made possible by the application of force. Microfluidic lab-on-a-chip approaches have proven helpful for parallelization, small sample volumes, reproducibility, and low cost. We report the successful combination of these two principles, forming a microfluidic molecular force assay and representing a novel use for the established MITOMI chip design. We present, characterize, validate and apply this integrated method. An alternative confocal fluorescence microscopy readout and analysis method is introduced and validated. In a multiplexing application, EcoRI binding is detected and characterized. This method paves the way for quantitative on-chip force measurements. It is suited for integration with DNA micro-spotting and in vitro expression of transcription factors to form a high-throughput chip for detailed DNA-protein interaction studies.

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Isolation of ORC6 , a Component of the Yeast Origin Recognition Complex by a One-Hybrid System

Cited by 398 publications

References 31 publications

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

Protein–DNA force assay in a microfluidic format

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