ASSEMBLY OF A CHROMOSOMAL REPLICATION MACHINE - 2 DNA-POLYMERASES, A CLAMP LOADER, AND SLIDING CLAMPS IN ONE HOLOENZYME PARTICLE .5. 4 DIFFERENT POLYMERASE CLAMP COMPLEXES ON DNA

Several different subassemblies of DNA polymerase III holoenzyme can b e purified from Escherichia coli. Toward the goal of understanding the functional significance of these subassemblies, we have used the gamm a complex clamp loader and the beta ring to assemble each different po lymerase onto DNA. Through use of radioactive labeled proteins, the su bunit structure of each resulting processive polymerase has been deter mined. Use of DNA polymerase III core, the gamma complex, and beta res ults in a core-beta complex on DNA; the gamma complex is not incorpora ted into the structure. The addition of tau to the assembly reaction t o form either core(1)-tau(2) or core(2)-tau(2) results in a more effic ient polymerase and more stabile association of core-tau beta on DNA, although the gamma complex still does not remain on DNA. The gamma com plex clamp loader was retained on DNA with the other subunits only if it was first assembled into the polymerase (Pol) III structure. The c lamp 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 b oth strands of a duplex chromosome. These findings extend those of an earlier study showing that distinctive polymerases can be assembled de pending on the presence or absence of tau (Maki, S., and Kornberg, A ( 1988) J. Biol. Chem. 263, 6561-6569). The significance of these distin ct polymerases in separate paths of DNA metabolism is discussed.