title: Quantitative proteomic analysis of yeast DNA replication proteins.
abstract: Chromatin is dynamically regulated, and proteomic analysis of its composition can provide important information about chromatin functional components. Many DNA replication proteins for example bind chromatin at specific times during the cell cycle. Proteomic investigation can also be used to characterize changes in chromatin composition in response to perturbations such as DNA damage, while useful information is obtained by testing the effects on chromatin composition of mutations in chromosome stability pathways. We have successfully used the method of stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis of normal and pathological changes to yeast chromatin. Here we describe this proteomic method for analyzing changes to Saccharomyces cerevisiae chromatin, illustrating the procedure with an analysis of the changes that occur in chromatin composition as cells progress from a G1 phase block (induced by alpha factor) into S phase (in the presence of DNA replication inhibitor hydroxyurea).
authors.affiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kubota
authors.forename: David A
authors.initials: DA
authors.lastname: Stead
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Hiraga
authors.forename: Sara
authors.initials: S
authors.lastname: ten Have
authors.forename: Anne D
authors.initials: AD
authors.lastname: Donaldson
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000302:isolation & purification / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006918:Hydroxyurea / Q000494:pharmacology; D007553:Isotope Labeling; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013104:Spheroplasts / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D002843:Chromatin; D004268:DNA-Binding Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D006918:Hydroxyurea