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 bacteriophage DNA polymerase holoenzyme to synthesize DNA processively on the leading strand of the replication fork for many minutes, while allowing an identical holoenzyme on the lagging strand to recycle from one Okazaki fragment to the next in less than 4 s. We use a perfect hairpin helix of 15 base pairs to mimic the encounter of the polymerase with the end of a previously synthesized Okazaki fragment. Polymerase dissociation is monitored during the stall at the hairpin helix by the addition of excess T4 gene 32 protein (SSB protein), which rapidly melts the helix and allows a stalled polymerase molecule to continue DNA synthesis. In the accompanying paper, we show that polymerase holoenzyme dissociation is slow (half-life of 2.5 min) when this enzyme is stalled by nucleotide omission (Hacker, K. J., and Alberts, B. M. (1994) J. Biol. Chem. 269, 24209-24220). In contrast, the holoenzyme dissociates with a half-life of 1 s after hitting the hairpin helix, a rate sufficient to allow efficient polymerase recycling on the lagging strand in vivo. We conclude that, upon completing each Okazaki fragment, the holoenzyme senses an encounter with duplex DNA and then switches to a state that rapidly dissociates.
Hacker
K J
KJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Alberts
B M
BM
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C022262:Okazaki fragments; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D004247:DNA; D004259:DNA-Directed DNA Polymerase