Abstract
Several different subassemblies of DNA polymerase III holoenzyme can be purified from Escherichia coli. Toward the goal of understanding the functional significance of these subassemblies, we have used the gamma complex clamp loader and the beta ring to assemble each different polymerase onto DNA. Through use of radioactive labeled proteins, the subunit structure of each resulting processive polymerase has been determined. Use of DNA polymerase III core, the gamma complex, and beta results in a core-beta complex on DNA; the gamma complex is not incorporated into the structure. The addition of tau to the assembly reaction to form either core1-tau 2 or core2-tau 2 results in a more efficient polymerase and more stabile association of core-tau beta on DNA, although the gamma complex still does not remain on DNA. The gamma complex clamp loader was retained on DNA with the other subunits only if it was first assembled into the polymerase (Pol) III* structure. The clamp loader within Pol III* appeared to be capable of loading two beta clamps onto DNA for both core polymerases within Pol III*, consistent with the hypothesis that one replicase can simultaneously replicate both strands of a duplex chromosome. These findings extend those of an earlier study showing that distinctive polymerases can be assembled depending on the presence or absence of tau (Maki, S., and Kornberg, A. (1988) J. Biol. Chem. 263, 6561-6569). The significance of these distinct polymerases in separate paths of DNA metabolism is discussed.
Highlights
From the Wicrobiology Department and the Hearst Research Foundation and the Woward Hughes Med ical Institute, Cornell University Medical College, New York, New York 10021
Toward the goal of understanding the functional significance of these subassemblies, w e have used the 'Y complex clamp loader and the p ring to assemble each different polymerase onto DNA
Use of DNA p olymerase III co r e, the 'Y complex, a n d /3 results in a core-B complex on DNA; the 'Y complex is not Incorporated into the structure
Summary
Of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader, and Sliding Clamps in One Holoenzyme Particle v. The cl amp loader w ithin Pol 111* appeared to be capable of loading two /3 cl amps o n t o DNA for both core polymer ase s within P ol 111*, co n sisten t with the hypothesis that one r eplicase can simultaneously r eplicate both strands of a duplex c h r o mosome. These findings extend those of an ear lier study showing that distinctive polyme r ases can b e a ssemb led depending on the presence or absence of T The costs of publication of this article were defrayed in part by the payment of page charges. § Present address: Dept. of Cell Biology, Harvard Medical School, Boston, MA 02115
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