Characterization of protein-DNA interactions during replication proces of plasmid pAG20 from Acetobacter aceti CCM 3620
Martin Babič 1
1 Univerzita Komenského v Bratislave, Prírodovedecká fakulta,, Bratislava, Slovenská republika
|Section:||Cellular metabolism, physiology, molecular biology and genetics|
Many plasmids are able to replicate in their original host and related species, and some of them have an extended host range which allows their establishment in several different bacterial species. At least two essential processes contribute to the host range phenotype: replication and partition Theplasmids became more interesting when scientist discovered that many plasmids contain genes responsible for antibiotic resistance and are responsible for the spread of antibiotic resistance among pathogen bacteria. Plasmids can give its host other kinds of resistance e.g. against heavy metals, bacteriocins, radiation and toxic anions. In this situation, the parasite host relation is more symbiotic, since the host gain some benefits. Many plasmids today are very important tools in gene technology and science e.g., as cloning vectors and expression vectors .
In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM 3620 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. 9 amino acids of the third a-helix (between 58 and 66 amino acids) play a key role in the Rep20-specific binding to theoriregulation region. Similarly, the region (9 amino acids between 49 and 57 amino acids) in the middle of the second and the third α-helices, containing the β-sheet structure, appeared to be also important for DNA binding
Targeted mutagenesis of the Rep20 protein revealed the importance of the third a-helix and 63Lys, specifically during DNA binding.The second, closely adjacentb-sheet also took part in this process in which 52Asn played a significant role to binding Rep20 to DNA .
 Babič, M., Rešková, Z., Bugala, J., Cimová, V., Grones, P.,Grones, J., (2014) The Rep20 Replication Initiator From the pAG20 Plasmid of Acetobacter aceti, Mol Biotechnol, 56:1–11.