IHF protein inhibits cleavage but not assembly of plasmid R388 relaxosomes.
Relaxosomes are specific nucleoprotein structures involved in DNA-processing reactions during bacterial conjugation. In this work, we present evidence indicating that plasmid R388 relaxosomes are composed of origin of transfer (oriT) DNA plus three proteins TrwC relaxase, TrwA nic-cleavage accessory protein and integration host factor (IHF), which acts as a regulatory protein. Protein IHF bound to two sites (ihfA and ihfB) in R388 oriT, as shown by gel retardation and DNase I footprinting analysis. IHF binding in vitro was found to inhibit nic-cleavage, but not TrwC binding to supercoiled DNA. However, no differences in the frequency of R388 conjugation were found between IHF- and IHF+ donor strains. In contrast, examination of plasmid DNA obtained from IHF- strains revealed that R388 was obtained mostly in relaxed form from these strains, whereas it was mostly supercoiled in IHF+ strains. Thus, IHF could have an inhibitory role in the nic-cleavage reaction in vivo. It can be speculated that triggering of conjugative DNA processing during R388 conjugation can be mediated by IHF release from oriT.
Moncalián
G
G
Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain.
Valle
M
M
Valpuesta
J M
JM
de la Cruz
F
F
D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D018983:DNA Footprinting; D004254:DNA Nucleotidyltransferases; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D019426:Integrases; D034982:Integration Host Factors; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011815:R Factors / Q000502:physiology*; D045522:Recombinases; D012097:Repressor Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004270:DNA, Circular; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; D009698:Nucleoproteins; D045522:Recombinases; D012097:Repressor Proteins; C119757:TrwA protein, E coli; D004254:DNA Nucleotidyltransferases; C110340:DNA relaxase; D019426:Integrases; C115006:integron integrase IntI1; D003850:Deoxyribonuclease I