title: The Rep20 replication initiator from the pAG20 plasmid of Acetobacter aceti.
abstract: In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5'-TCCAAATTTGGAT'-3') and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and ⁶³Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which ⁵²Asn played a significant role.
authors.affiliation: Department of Molecular Biology, Comenius University, Mlynská dolina B-2, 842 15, Bratislava 4, Slovak Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Babič
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Rešková
authors.forename: Juraj
authors.initials: J
authors.lastname: Bugala
authors.forename: Viera
authors.initials: V
authors.lastname: Cimová
authors.forename: Peter
authors.initials: P
authors.lastname: Grones
authors.forename: Jozef
authors.initials: J
authors.lastname: Grones
mesh_terms: D000091:Acetobacter / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008958:Models, Molecular; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D001216:Asparagine