THE RAPID DISSOCIATION OF THE T4 DNA-POLYMERASE HOLOENZYME WHEN STOPPED BY A DNA HAIRPIN HELIX - A MODEL FOR POLYMERASE RELEASE FOLLOWING THE TERMINATION OF EACH OKAZAKI FRAGMENT

We have examined the molecular mechanism that enables the T4 bacteriop hage DNA polymerase holoenzyme to synthesize DNA processively on the l eading strand of the replication fork for many minutes, while allowing an identical holoenzyme on the lagging strand to recycle from one Oka zaki fragment to the next in less than 4 s. We use a perfect hairpin h elix of 15 base pairs to mimic the encounter of the polymerase with th e end of a previously synthesized Okazaki fragment. Polymerase dissoci ation is monitored during the stall at the hairpin helix by the additi on of excess T4 gene 32 protein (SSB protein), which rapidly melts the helix and allows a stalled polymerase molecule to continue DNA synthe sis. In the accompanying paper, we show that polymerase holoenzyme dis sociation is slow (half-life of 2.5 min) when this enzyme is stalled b y nucleotide omission (Hacker, K. J., and Alberts, B. M. (1994) J. Bio l. Chem. 269, 24209-24220). In contrast, the holoenzyme dissociates wi th a half-life of 1 s after hitting the hairpin helix, a rate sufficie nt to allow efficient polymerase recycling on the lagging strand in vi vo. We conclude that, upon completing each Okazaki fragment, the holoe nzyme senses an encounter with duplex DNA and then switches to a state that rapidly dissociates.