Modification of the plasmid initiator protein RepC active site during replication.
pT181 is a Staphylococcus aureus rolling circle replicating plasmid whose copy number is controlled by regulating the synthesis and activity of the initiator protein, RepC*. The RepC* dimer is modified during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC. To purify RepC, RepC was expressed in S. aureus as a fusion protein with a polyhistidine tail. The histidine-tagged RepC retains its initiation and topoisomerase activities in vitro. His-tagged RepC/RepC and RepC/RepC* were purified in a two-step procedure. Peptide mapping, mass spectrometric analysis and protein sequencing of purified RepC and RepC* were carried out, and both proteins appeared identical, except that the peptide containing the RepC active site tyrosine used in nicking activity was absent when the purified RepC* sample was analyzed. The absence of the active site in RepC* suggests that this site was modified during replication. The results provide the first direct biochemical evidence that RepC* is a modified form of RepC, and support a model in which RepC replication of pT181 leaves RepC with an oligonucleotide blocking the active site of one of its subunits.
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Skirball Institute/NYU Medical Center New York, USA.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D003001:Cloning, Molecular; D004261:DNA Replication / Q000502:physiology*; D005798:Genes, Bacterial / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational; D013211:Staphylococcus aureus / Q000235:genetics
D001426:Bacterial Proteins; D010455:Peptides; C043650:RepC protein, Staphylococcus aureus; D006639:Histidine