Mouse primase initiation sites in the origin region of simian virus 40

The sites of initiation of DNA synthesis by purified mouse DNA primase in the origin-of-replication region of simian virus 40 (SV40) were examined. Using as template the separated strands of a cloned fragment of SV40 approximately equal to 300 base pairs (bp) long that includes the origin, we observed specific sites of initiation on the two strands. On the early strand that is the template for early mRNA synthesis, the primary starts are at four positions within 10 nucleotides of each other around nucleotide 5215 and an additional site around nucleotide 5147 that is used at one-sixth the frequency of the major sites. The major start sites on the early strand are within the 65-bp minimal origin of replication and lie between tumor antigen binding sites I and II. On the late strand that is the template for late mRNA synthesis, six major initiation sites were observed, each within the 3' C-C-C-G-C-C 5' sequence in the template that is repeated twice within each of the three 21-bp repeats that lie adjacent to the minimal origin, on its late side. A 6-bp deletion in the 65-bp minimal origin that eliminates its function as an origin reduced the major initiations around nucleotide 5215 on the early strand by 90% but did not affect initiations at the minor start site on the early strand or initiations on the late strand. Mouse DNA primase is able to recognize specific regions on the SV40 DNA. Those on the early strand are within the minimal origin of replication and those on the late strand are within the 21-bp repeat region necessary for maximum replication.