Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces

Binding Modes of the Initiator and Inhibitor Forms of the Replication Protein π to the γ ori Iteron of Plasmid R6K

“…Complementing this, hyperreplicative (i.e. copy-up) variants tend to be more susceptible than WT to guanidine HCl treatment (suggesting dimer instability) and tend to show more monomer complex in EMSA, whereas inactive initiator variants produce predominantly dimer complex (9,12,13,15,20) (this work). Recently, Bastia and co-workers (9) combined Sephadex G-75 column filtration (for molecular weight determination), guanidine HCl challenge, mutant analysis, and EMSA in a single set of experiments.…”

“…To identify the components of these complexes, a technique was employed where full-length and shorter variants were reconstituted into heterodimers prior to DNA binding. Comparative analyses of the resultant banding patterns (in EMSA) suggested that, for WT , the faster migrating complex contains bound monomers of the protein, and the slower migrating complex contains bound dimers (12,14,15). Moreover, the binding proficiency of heterodimers in which one variant lacked the C-terminal DNA binding domain suggested that only one subunit of a dimer makes specific contact with the iteron DNA (15).…”

“…In EMSA, increasing guanidine HCl concentrations led to a loss of the slower migrating "dimer" band and an increase in the faster migrating, "monomer" band (12,15). Treatment with guanidine HCl or urea has also been shown to activate a Rep protein (RepA), as has treatment with chaperones (e.g.…”

“…In the work presented here, we used His-tagged wild type (His-⅐WT) and previously characterized variants of the protein with altered monomer/dimer ratios (His-⅐P106L/F107S (14,15) and His-⅐M36A/M38A (13)) to study iteron/ interactions. Each variant differs from the other in several replicationrelated functional properties such as iteron binding and the ability to facilitate "open complex" formation.…”

“…Each variant differs from the other in several replicationrelated functional properties such as iteron binding and the ability to facilitate "open complex" formation. His-⅐P106L/ F107S is a copy-up variant; it stimulates increased replication compared with WT , apparently by elevating the fraction of monomers relative to the low monomer level observed in the WT protein (14,15). The variant His-⅐M36A/M38A fails to support replication, and the highly purified protein does not possess strand-opening activity in vitro (13).…”