Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates

Abstract

Replication complexes were reconstituted using the eight purified bacteriophage T4 replication proteins and synthetic circular 70-, 120- or 240-nt DNA substrates annealed to a leading-strand primer. To differentiate leading strands from lagging strands, the circular parts of the substrates lacked dCMP; thus, no dCTP was required for leading-strand synthesis and no dGTP for lagging-strand synthesis. The size of the substrates was crucial, the longer substrates supporting much more DNA synthesis. Leading and lagging strands were synthesized in a coupled manner. Specifically targeting leading-strand synthesis by decreasing the concentration of dGTP decreased the rate of extension of leading strands. However, blocking lagging-strand synthesis by lowering the dCTP concentration, by omitting dCTP altogether, by adding ddCTP, or with a single abasic site had no immediate effect on the rate of extension of leading strands.