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dc.contributor.authorBaker, Richard E.
dc.contributor.authorFitzgerald-Hayes, Molly
dc.contributor.authorOâBrien, Timothy C.
dc.date2022-08-11T08:10:05.000
dc.date.accessioned2022-08-23T16:54:37Z
dc.date.available2022-08-23T16:54:37Z
dc.date.issued1989-06-25
dc.date.submitted2008-08-15
dc.identifier.citationJ Biol Chem. 1989 Jun 25;264(18):10843-50.
dc.identifier.issn0021-9258 (Print)
dc.identifier.pmid2543684
dc.identifier.urihttp://hdl.handle.net/20.500.14038/42541
dc.description.abstractCP1 is a yeast protein which binds to the highly conserved DNA element I (CDEI) of yeast centromeres. We have purified CP1 to near homogeneity; it is comprised of a single polypeptide of molecular weight 58,400. When bound to yeast CEN3 DNA, CP1 protects a 12-15-base pair region centered over CDEI. Methylation interference experiments show that methylations of residues located outside of the 8-base pair CDEI sequence have no detectable effect on CP1 binding, suggesting that the DNA sequences important for CP1 recognition are confined to the CDEI octanucleotide. The equilibrium constant for CP1 binding to CEN3 DNA is relatively low, 3 x 10(8) M-1. Using a novel method to determine relative DNA binding constants, we analyzed the effect of CDEI mutations on CP1 binding. A C to T point mutation at position 5 (CO1) reduces the equilibrium constant about 35-fold, while the insertion of an additional T at this position (CAT) reduces the equilibrium constant 1,400-fold. The effect of these mutations on mitotic centromere function in vivo was assessed using a plasmid stability assay. While the CO1 mutation had a slight effect, the CAT mutation significantly impaired function, implying that CP1 binding is required for the optimal mitotic function of yeast centromeres.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=2543684&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://www.jbc.org/content/264/18/10843.full.pdf+html
dc.subject3',5'-Cyclic-AMP Phosphodiesterases
dc.subjectBase Sequence
dc.subjectCentromere
dc.subjectChromosomes
dc.subjectDNA, Fungal
dc.subjectDNA-Binding Proteins
dc.subjectElectrophoresis, Polyacrylamide Gel
dc.subjectKinetics
dc.subjectMolecular Sequence Data
dc.subjectPlasmids
dc.subjectProtein Binding
dc.subjectRestriction Mapping
dc.subjectSaccharomyces cerevisiae
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titlePurification of the yeast centromere binding protein CP1 and a mutational analysis of its binding site
dc.typeJournal Article
dc.source.journaltitleThe Journal of biological chemistry
dc.source.volume264
dc.source.issue18
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/880
dc.identifier.contextkey579769
html.description.abstract<p>CP1 is a yeast protein which binds to the highly conserved DNA element I (CDEI) of yeast centromeres. We have purified CP1 to near homogeneity; it is comprised of a single polypeptide of molecular weight 58,400. When bound to yeast CEN3 DNA, CP1 protects a 12-15-base pair region centered over CDEI. Methylation interference experiments show that methylations of residues located outside of the 8-base pair CDEI sequence have no detectable effect on CP1 binding, suggesting that the DNA sequences important for CP1 recognition are confined to the CDEI octanucleotide. The equilibrium constant for CP1 binding to CEN3 DNA is relatively low, 3 x 10(8) M-1. Using a novel method to determine relative DNA binding constants, we analyzed the effect of CDEI mutations on CP1 binding. A C to T point mutation at position 5 (CO1) reduces the equilibrium constant about 35-fold, while the insertion of an additional T at this position (CAT) reduces the equilibrium constant 1,400-fold. The effect of these mutations on mitotic centromere function in vivo was assessed using a plasmid stability assay. While the CO1 mutation had a slight effect, the CAT mutation significantly impaired function, implying that CP1 binding is required for the optimal mitotic function of yeast centromeres.</p>
dc.identifier.submissionpathoapubs/880
dc.contributor.departmentDepartment of Molecular Genetics and Microbiology
dc.source.pages10843-50


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