Initiation of Deoxyribonucleic Acid Synthesis

Replication of M13 viral strands with an enzyme preparation from Escherichia coli produces a duplex (RF–II) with a nearly full length complementary strand (Wickner, W., Brutlag, D., Schekman, R., Kornberg, A., (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 965–969). The gap in this strand relative to the viral circle has a unique location as judged by the following criteria: (a) a Haemophilus influenzae restriction enzyme preparation breaks the RF–II at one specific site near the gap, cleaving the complementary strand into one large and one small fragment; (b) a Haemophilus parainfluenzae restriction enzyme preparation cleaves the RF–I into at least nine fragments, one of which is missing from the RF–II and is restored when the gap is filled by DNA polymerase. These newly added deoxyribonucleotides are primarily associated with the small fragment after cleavage by H. influenzae enzyme, establishing the proximity of this cleavage site near the 3' end of the complementary strand; (c) pyrimidine tracts from the repaired region (and representing the gap) have a simple and distinctive composition, compared with the full complementary strand; and (d) cleavage of the RF–II at the gap by the single-strand-specific endonuclease S1 from Aspergillus oryzae yields linear molecules which after melting and re-annealing from linear rather than circular duplexes, demonstrating that the gap is at only one place relative to the viral strand.