SYNTHESIS ON DOUBLE-STRANDED CIRCULAR DNA CATALYZED BY THE DNA-POLYMERASE ALPHA-DNA PRIMASE COMPLEX FROM SILKWORM CELLS

A replicative complex containing DNA polymerase alpha and DNA primase from cells of the silkworm Bombyx mori is able to initiate DNA synthesis on circular, super-coiled pBR322 or pUC19 DNA in the presence of NTPs. During initiation on these circular DNAs, the primase component of the complex synthesizes oligoribonucleotides approximately 10 residues long that serve as primers for subsequent elongation by the DNA polymerase-alpha. The nascent Okazaki fragments vary from tens to hundreds of nucleotides in length and tend to dissociate from the double-stranded templates because of thermal fluctuations in the secondary and tertiary structures of the circular DNA molecules. Agarose gel electrophoresis of the products of synthesis revealed the existence of a topological form of pUC19 DNA that migrates faster than the negatively supercoiled topoisomer (form I) and serves as an efficient substrate for NTP-dependent DNA synthesis catalyzed by the silkworm polymerase-primase complex.