DNA polymerase III holoenzyme

DNA polymerase III can polymerize DNA, but with a much lower processivity than one would expect for the organized replication of an entire chromosome. The necessary increase in processivity is provided by the addition of the J8 subunits, four of which complete the DNA polymerase III holoenzyme. The J3 subunits associate in pairs to form donut-shaped structures that encircle the DNA and act like clamps (Fig. 25-10b). Each dimer associates with a core subassembly of polymerase III (one dimeric clamp per core subassembly) and slides along the DNA as replication proceeds. The J8 sliding clamp prevents the dissociation of DNA polymerase III from DNA, dramatically increasing processivity—to greater than 500,000 (Table 25-1). 

DNA polymerase III is also a multifunctional enzyme. It resembles DNA polymerase I in catalytic properties however, there are slight differences with respect to the type of template primer preferred for DNA synthesis as well as the preferred substrates for the two exonuclease activities. It contains many polypeptide subunits (a, e, 6, t, y, 8. y, ip and B)- The complex containing all subunits (Mr 900,000) is called the DNA polymerase III holoenzyme, while that comprising just a, e and 8 exhibits the polymerase activity and is referred to as the core enzyme. The holoenzyme carries out most of the DNA synthesis at the replication fork in vivo. 

The RNA primer must be removed and replaced by DNA. This is accomplished as follows The DNA polymerase III holoenzyme ceases its action (without dissociation from the DNA) when it reaches the 5 end of the RNA primer, and DNA polymerase I takes over. A feature of DNA polymerase I, in contrast to other DNA polymerases, is its ability to effect replication at a nick. It doesn t matter if the polynucleotide ahead of the nick is DNA or RNA. Referring to Fig. 16-12, DNA polymerase I binds at the solid left arrowhead and continues to extend the DNA chain. At the same time, it removes the RNA primer through its 5 —>3 exonuclease activity. The overall effect is for it to replace the RNA by DNA, and this continues until the nick has shifted beyond the RNA section. This is an example of nick translation. Once the nick is bounded by DNA, it is sealed. 

The 3 —>5 exonuclease activity of DNA polymerase I, at least, functions to proofread for such mistakes. After the incorrect base is incorporated, it will not remain hydrogen-bonded to the tautomeric base in the template once the latter returns, almost immediately, to its more stable form. The 3 — 5 exonuclease activity shows a strong preference for a frayed or non-hydrogen-bonded end and removes the misincorporated nucleotide before chain growth proceeds further. DNA polymerase III holoenzyme also has the potential to proofread by the same mechanism. 

The elongation phase of DNA replication in bacteria has been seen to involve many enzymes and proteins, and some are associated with discrete functional complexes, such as the DNA polymerase III holoenzyme. Initiation of replication also uses several proteins, and mutations in their genes have been very helpful in identifying these proteins. 

High-fidelity chromosomal replication in E. coli is executed by a multicomponent complex referred to as DNA polymerase III holoenzyme (see Fig. 4a) (18-21). Pol 111 holoenzyme consists of three main subcomponents Pol 111 core, 3-clamp, and y-complex clamp-loader. Pol 111 core is the replicative DNA polymerase that consists of three subunits (a, e, 0) a exhibits DNA polymerase activity, e performs 3 -5 exonuclease activity necessary for proofreading, and the function of 0 is currently unclear. 

McHenry CS. DNA polymerase III holoenzyme. Components, structure, and mechanism of a true replicative complex. J. Biol. Chem. 1991 266 19127-19130. 

O Donnell M, Jeruzalmi D, Kuriyan J. Clamp loader structure predicts the architecture of DNA polymerase III holoenzyme and RFC. Curr. Biol. 2001 11 935-946. 

Glover BP, McHenry CS. The chi psi subunits of DNA polymerase III holoenzyme bind to single-stranded DNA-binding protein (SSB) and facilitate replication of an SSB-coated template. J. Biol. Chem. 1998 273 23476-23484. 

DNA polymerase III holoenzyme, an asymmetric dimer. The discontinuous assembly of the lagging strand enables 5 —>... 

Molecular motors in replication, (a) How fast does template DNA spin (expressed in revolutions per second) at an E. coli replication fork (b) What is the velocity of movement (in micrometers per second) of DNA polymerase III holoenzyme relative to the template ... 

Kong, X. P., Onrust, R., O Donnell, M., and Kuriyan, J. (1992) Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme a sliding DNA clamp. Cell 69, 425 137. 

Kelman, Z. and O Donnell, M. (1995) DNA polymerase III holoenzyme structure and function of a chromosomal replicating machine. Annu. Rev. Biochem., 64,171-200. 

Tomer, G., Reuven, N.B., and Iivneh, Z. (1998) The (1 subunit sliding DNA damp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme. Proc. Natl. Acad. Sci. USA, 95, 14106-14111. 

DNA polymerase vs. RNA polymerase - Vmax (see here) for the DNA polymerase III holoenzyme,... 

Replicative DNA chain growth is rapid but occurs at few sites, whereas transcription is much slower, but occurs at many sites. The result is that far more RNA accumulates in the cell than DNA. Like the DNA polymerase III holoenzyme, the action of RNA polymerase is highly processive. Once transcription of a gene has been initiated, RNA polymerase rarely, if ever, dissociates from the template until the specific... 

It may be the principal leading strand polymerase. Requires a protein called proliferating cell nuclear antigen (PCNA) to carry out highly processive DNA synthesis in vitro. PCNA functions like the clamp of E. coli DNA Polymerase III holoenzyme. 

DNA polymerase III holoenzyme is a complex of several proteins. The polC gene encodes a single polypeptide chain of Mr of about 130,000. The protein has an intrinsic polymerase activity, but it is quite low. In cells, however, the PolC protein functions as part of a multiprotein aggregate called the DNA polymerase III holoenzyme. Figure 24.19 shows that the holoenzyme contains 10 different polypeptide chains, each identified with a Greek letter. The functions of these units are summarized as follows ... 

Figure 24.19 Subunit structure of the E. coli DNA polymerase III holoenzyme. 

DNA polymerase III holoenzyme extends both leading and lagging strands. 

Structure and Mechanism of DNA Polymerases The DNA Polymerase III Holoenzyme Eukaryotic DNA Polymerases (Table 24.3)... 

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