title: Probing the N-terminus of ParB using cysteine-scanning mutagenesis and thiol modification.
abstract: Plasmid partition systems require site-specific DNA binding proteins to recognize the plasmid partition site, or centromere. When bound to the centromere, these proteins, typically called ParB, interact with the ParA ATPases, which in turn promote the proper positioning of plasmids prior to cell division. P1 ParB is a typical member of a major class of ParB-like proteins that are dimeric helix-turn-helix DNA binding proteins. The N-terminus of ParB contains the region that interacts with ParA and with itself, but it has been difficult to study because this region of the protein is flexible in solution. Here we describe the use of cysteine-scanning mutagenesis and thiol modification of the N-terminus of ParB to create tools to probe the interactions of ParB with itself, with ParA and with DNA. We introduce twelve single-cysteine substitutions across the N-terminus of ParB and show that most do not compromise the function of ParB and that none completely inactivate the protein in vivo. We test three of these ParB variants in vitro and show that they do not alter ParB function, measured by its ability to stimulate ParA ATPase activity and its site-specific DNA binding activity. We discuss that this approach will be generally applicable to the ParB-like proteins in this class of partition systems because of their natural low content of cysteines, and because our evidence suggests that many residues in the N-terminus are amenable to substitution by cysteine.
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors: Anthony G
authors: AG
authors: Vecchiarelli
authors: Barbara E
authors: BE
authors: Funnell
mesh_terms: D016375:Antisense Elements (Genetics) / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D019915:DNA Primase / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D016375:Antisense Elements (Genetics); D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C074125:RepB' protein, E coli; D019915:DNA Primase; D012319:RNA Polymerase II; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; C121791:dnaG protein, E coli; D003545:Cysteine