OPEN STRANDS ADJACENT TO ITERONS PROMOTE THE BINDING OF THE REPLICATION INITIATOR PROTEIN (REP) OF PSC101 TO THE UNIT SEQUENCE OF THE ITERONS IN-VITRO

The purified dimeric form of the Rep protein, a replication initiator protein of the plasmid pSC101, has a low affinity for repeated sequenc es, iterons, in the replication origin of the plasmid, and higher affi nities for two inverted repeats in the operator region of the rep gene resulting in its functioning as an autorepressor. Studies of binding to various synthetic DNA have established that Rep can bind to duplex iteron-sequence carrying open (non-complementary) strands at one end p roximal to the rep gene. Open strands at the opposite end of the itero n have no effect on Rep-binding. One open strand seems to be required in a sequence-specific fashion. A randomly sequenced duplex DNA with t he open strands cannot bind to Rep but can function as a significant c ompetitor. This suggests that Rep has some affinity for the open stran ds and forms a stable complex with the adjacent iteron. The mutated Re p protein, Rep1, which causes an increase in the plasmid copy number i n vivo, has equally high affinity for the iteron with the open strands as wild type Rep, though it has a lower affinity for the inverted rep eats than the wild type. The Rep dimer might bind to these DNA sequenc es with different modes.