MULTIPLE PATHWAYS OF COPY CONTROL OF GAMMA-REPLICON OF R6K - MECHANISMS BOTH DEPENDENT ON AND INDEPENDENT OF COOPERATIVITY OF INTERACTION OF PI-PROTEIN WITH DNA AFFECT THE COPY NUMBER

The ability of a replication initiator protein to promote intermolecular pairing of two replication origins resulting in the turning off of the origin pair has been called handcuffing. We have endeavored to test the validity of the handcuffing model by isolating two mutant forms of the pi initiator protein of R6K that elicit high copy number phenotype. We have discovered that one mutant called pi 113 yielded a 3.6-fold increase in copy number of a gamma replicon with a concomitant impairment of its ability to loop DNA and to pair binding sites (iterons) in comparison with normal pi, thus supporting the handcuffing model. A second mutant called pi 108, on the other hand, elicited a 3-fold increase in copy number without showing any measurable loss in its ability to loop and pair gamma iterons. Both mutant forms of the wild-type pi protein showed no detectable differences in their affinity of binding to the gamma iterons. Thus, the phenotype of pi 108 is consistent with the proposition that copy number control involves macromolecular interactions other than cooperativity at a distance of pi or interaction of pi with the primary binding sites at gamma. Taken together, the results are consistent with the notion that pi-mediated handcuffing is a mechanism, but not the only mechanism, of copy control In R6K. Interaction of pi with host proteins is likely to provide additional facets of the copy control mechanism.