COORDINATED LEADING AND LAGGING-STRAND DNA-SYNTHESIS ON A MINICIRCULAR TEMPLATE

The coordinated synthesis of both leading and lagging DNA strands is t hought to involve a dimeric DNA polymerase and a looping of the laggin g strand so that both strands can be synthesized in the same direction . We have constructed a minicircle with a replication fork that permit s an assessment of the stoichiometry of the proteins and a measurement of the synthesis of each strand. The replisome consisting of bacterio phage T7 DNA polymerase, helicase, primase, and single-stranded DNA-bi nding protein mediates coordinated replication. The criteria for coord ination are fulfilled: (1) a replication loop is formed, (2) leading a nd lagging strand synthesis are coupled, (3) the lagging strand polyme rase recycles from one Okazaki fragment to another, and (4) the length of Okazaki fragments is regulated. T7 single-stranded DNA-binding pro tein is essential for coordination.