Function
RepA_N is a highly conserved replication initiation protein that mediates replication of plasmids containing multidrug resistance genes. The protein studied here is purified from Staphylococcus aureus in particular. RepA_N proteins are encoded by plasmids from other Gram-positive bacteria.
The protein forms a tetramer in the cell, formed first by two dimers. Each dimer is a functional group that faces opposite directions. The dimer recognize iteron boxes 20-25 bp in length, which may occur multiple times on a multiresistance plasmid. The protein may initiate replication in two plasmids at once, by a mechanism called "RepA-RepA handcuffing." The binding action deforms DNA by bending, which may assist in melting during replication. Multiple RepA proteins may interact during DNA binding by cooperative action.
Current research suggests that RepA_N is the sole facilitator in replicon assembly, not needing any cofactors or coenzymes, except a RepA C-terminal domain. This leads to multiresistance plasmids requiring minimal input from the cell to maintain themselves.
Relevance
Multiresistance plasmids are a contributing factor to bacterial resistance to antibiotics. The rapidity with which bacteria have evolved these features has posed a threat to medicine. Given that RepA_N is the sole mediator for the propagation of these plasmids, further study may reveal a tool to prevent the production of drug resistance proteins in bacteria.
Structural highlights
The subunits of the protein consist of a central . A helix-strand-helix occupies and a helix-loop motif occupies the . The terminal structures allow the subunit to form its quaternary structure, and the central winged HTH is the functional region of the protein.
The helicies of the protein , with the wings . The wing is inserted by Gly101 at the tip, allowing a deeper insertion into the minor groove than otherwise possible. Leu102 and Asn 103 interact with bases as well. assist in association with DNA via hydrogen bonding in the case of Lys79 and Glu80, and van der Waals forces in the case of Thr83. No major conformational change occurs upon binding, but rather the DNA is bent. The DNA iteron is typically centered about a extended AT tract.
