Active Allocation Mechanism For Parb Protein. Leifsonia Xyli Subsp.cynodontis Plxc100 Plasmid

The process that genomes must be equitably partitioned to daughter cells is essential to ensure faithful transmission of duplicated genetic material to next generation. The molecular events that direct accurate segregation of eukaryotic chromosomes were well documented. However, the segregation of plasmids and chromosomes in prokaryote was remained to understand. Up to now, two trans-acting proteins and a cis-acting centromere-like site are involved to this process in prokaryote, which was called active partition mechanism. One of the two proteins is a typical ATPase, and the other is a DNA binding protein that can bind centromere-like site and interact with the ATPase to form partition complex that mediates intracellular genome segregation. Most researches focus on the gram-negative bacteria, the knowledge of gram-positive is very limited. Two proteins,. ParA and ParB, and centromere-like site parS of cryptic plasmid pLXC100 from gram-positive bacterium Leifsonia xyli subsp. cynodontis involving to the plasmid segregation have been identified. It is shown that ParB is a DNA-binding protein, and ParA is a typical walk-type ATPase whose activity is influenced by ParB and parS. It is also found that ParA, ParB and parS can form partion complex. This is the first example of such a completed partition cassettes founded in gram-positive bacterium plasmid,which belongs to Ib type of plasmid active partition mechanism.Based on the former achievement,this project focused on characterizing ParB protein of par locus of plasmid pLXC100 from gram-positive bacterium through many kinds of biochemistry technology.Using of IMPACT-TWIN protein purifiction system,we got native ParB protein without an affinity tag that may alter its characteristics. EMSA,DnaseI footprinting and SPR analysis confirm that ParB protein has DNA binding activity and the binding kinetics parameter was got.Based on the prediction of its secondary structure,we cloned and expressed five mutants to explore its amino acid binding to DNA and its secondary structure using EGS chemistry cross-linking ,HPLC and CD technics.The results shew K70 was very important for ParB to keep its DNA binding activity. All the results of this project may enriches the research of Ib type of plasmid active partition mechanism,and also propels the developing research about the segregation of plasmids and chromosomes in prokaryote.