| My research project involved the role of two proteins, ArgR and PepA, which act as accessory factors in the ColE1 cer recombination system from the gram negative bacteria Escherichia coli. These two proteins, in addition to the tyrosine recombinases XerC and XerD, catalyze a site-specific recombination event at the cer sequence which converts unstable multimeric forms of ColE1 into more stable monomers. Our study mainly focused on the C-terminal end of the ArgR. This region of the ArgR protein possesses a structural and amino acid sequence similarity with the AhrC protein from Bacillus subtilis. Moreover, AhrC, the Arginine repressor of this bacterium, is able to complement Escherichia coli mutants deficient in ArgR. The C-terminal regions of these proteins, display a very high region of similarity. Previous work from our laboratory has shown that ArgR mutants with mutations in this region, especially the mutants ArgR149, a truncated 149 amino acids form of ArgR, and ArgR5aa, a form containing a five amino acid insertion in the C-terminal part, lost the ability to perform a recombination reaction at two cer sites in the plasmid pCS210. Despite this defect in promoting cer recombination, the mutants were still able to bind specifically to DNA, and to repress an argA : : lacZ genetic fusion. In this work, both wild type and mutant ArgR proteins were overexpressed as 6-histidine fusion proteins. Crosslinking analysis showed that both wild type and ArgR5aa efficiently formed hexamers in vitro, while ArgR149 formed lower molecular weight multimers. Truncated forms of ArgR that were 150 amino acids or longer, were able to support cer recombination. The substitution mutants between positions 149 to 151 all showed that single amino acid substitutions at this region had little effect on cer recombination. Protein-protein crosslinking experiments showed that wild type and mutant forms of ArgR, were able to interact with and the other accessory protein involved in cer recombination, PepA. In vitro recombination experiments using wild type and mutant forms of ArgR, combined with purified PepA, XerC and XerD showed that the ArgR149 mutant did not support recombination, but the ArgR5aa mutant did promote Holliday junction formation, raising the possibility that these two mutants interact differently with the Xer recombination machinery. Topology experiments showed that after adding topoisomerase 1, PepA is able to protect DNA from topoisomerase 1, and prevent ArgRWt binding to DNA. The two mutants ArgR149 and ArgR5aa are protecting DNA with more supercoiling. When PepA is added, migration profiles with the two mutants showed a binding problem with PepA. Other experiments involving the LEL triplet (leucine-glutamic acid-leucine) and amino-acids around it should be done in order to know the existence of a possible binding site for PepA.;Key-words: ArgR, PepA, ArgR C-terminus part, recombination, multimers, Holliday junction, protein to protein interaction. |
