PMID: 8961563Dec 1, 1996Paper

Modification of the plasmid initiator protein RepC active site during replication

FEMS Microbiology Letters
A Rasooly, R S Rasooly

Abstract

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.

References

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Feb 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·C C Kumar, R P Novick
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Apr 1, 1994·Journal of Bacteriology·A RasoolyR P Novick

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Citations

Dec 24, 1997·Trends in Microbiology·A Rasooly, R S Rasooly
Jun 10, 1998·Microbiology and Molecular Biology Reviews : MMBR·G del SolarR Díaz-Orejas

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