Replication DNA polymerase

Like bacteria, eukaryotes have several types of DNA polymerases. Some have been linked to particular functions, such as the replication of mitochondrial DNA. The replication of nuclear chromosomes involves DNA polymerase a, in association with DNA polymerase S. DNA polymerase a is typically a multisubunit enzyme with similar structure and properties in all eukaryotic cells. One subunit has a primase activity, and the largest subunit (Afr -180,000) contains the polymerization activity. However, this polymerase has no proofreading 3 —>5 exonuclease activity, making it unsuitable for high-fidelity DNA replication. DNA polymerase a is believed to function only in the synthesis of short primers (containing either RNA or DNA) for Okazaki fragments on the lagging strand. These primers... 

As an example, the cost-selectivity equation is plotted in Figure 13.9 for / = ff = 104 or 105, the measured discrimination factors for base pairing. Significant increases in specificity are reached only when the cost becomes appreciable. The measured cost for the replicational DNA polymerase of E. coli in vitro is 6 to 13%, depending on the dNTP concerned.53 This must be at the limits of the tolerable. Under these conditions, S 107 to 109 these values are too low... 

Eukaryotic replicative DNA polymerase a, 8, e, archaebacterial DNA polymerases, viral DNA polymerases, DNA polymerases encoded by mitochondrial plasmids of various fungi and plants, and some bacteriophage... 

Hogg, M., Wallace, S. S., and Doublie, S. (2004). Crystallographic snapshots of a replicative DNA polymerase encountering an abasic site. EMBOJ. 23, 1483-1493. 

Woodgate, R. (1999). A plethora of lesion-replicating DNA polymerases. Genes Dev. 13, 2191-2195. 

AutoDock6 of 25ID in DNA polymerase III a-subunit A homology model was used to dock a potent inhibitor of bacterial replicative DNA polymerase 25ID, which suggested active site residues involved in the interaction Chhabra etal. (88)... 

DNA replication at the site opposite to the adduct. In addition, DNA polymerase P was able to elongate the arrested replication products of the other three DNA polymerases in their presence, thus showing its capacity to successfully compete with them at a stalled replication complex. These results suggest that only DNA polymerase P, possibly because of its distributive mode of action and simple subunit composition, can productively associate with the primer/template junction formed at the base preceding the d(GpG) adduct and continue DNA elongation in a reaction which includes the replicative DNA polymerases. 

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. 

Bailey S, Wing RA, Steitz TA. The structure of T. aquaticus DNA polymerase III is distinct from eukaryotic replicative DNA polymerases. Cell 2006 126 893-904. 

Earners MH, Georgescu RE, Lee SG, O Donnell M, Kuriyan J. Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III. Cell 2006 126 881-892. 

Yuzhakov A, Kelman Z, O Donnell M. Trading places on DNA-a three-point switch underlies primer handoff from primase to the replicative DNA polymerase. Cell 1999 96 153-163. 

The tendency of these trinucleotide repeats to expand is explained by the formation of alternative structures in DNA replication (Figure 28.35). Part of the array within a template strand can loop out without disrupting base-pairing outside this region. In replication, DNA polymerase extends this strand through the remainder of the array by a poorly understood mechanism, leading to an increase in the number of copies of the trinucleotide sequence. 

The replicative DNA polymerases themselves are able to correct many DNA mismatches produced in the course of replication. For example, the subunit of E. co/i DNA polymerase III functions as a 3 -to-5 exonuclease. This domain removes mismatched nucleotides from the 3 end of DNA by hydrolysis. How does the enzyme sense whether a newly added base is correct As a new strand of DNA is synthesized, it is proojread. If an incorrect base is inserted, then DNA synthesis slows down owing to the difficulty of threading a non-Watson-Crick base pair into the polymerase. In addition, the mismatched base is weakly bound and therefore able to fluctuate in position. The delay from the slowdown allows time for these fluctuations to take the newly synthesized strand out of the polymerase active site and into the exonuclease active site (Figure 28.41). There, the DNA is degraded, one nucleotide at a time, until it moves back into the polymerase active site and synthesis continues. 

Wong SW, Wahl AF, Yuan P-M, Arai N, Pearson BE, Arai K-I, Korn D, Hunkapiller MW, Wang TS-E (1988) Human DNA polymerase a gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J 7 37-47... 

Depurinating adducts leave behind an abasic site on the DNA. Replicative DNA polymerase will insert an A opposite an abasic site so that depurination provides a straightforward route to either G — T or A —> T transversions when the daughter strand is replicated [34], As radical cation depurinating adducts result in N-glyco-sidic bond cleavage, repair of the abasic site becomes important. Apurinic/apyri-midinic endonuclease creates a strand break at the site of the lesion, and the gap is then repaired by phosphodiesterase, DNA polymerase, and DNA ligase [35], as discussed in Chapter 11. 

Avkin, S., Adar, S., Blander, G., and Livneh, Z. (2002) Quantitative measurement of translesion replication in human cells evidence for bypass of abasic sites by a replicative DNA polymerase. Proc. Natl. Acad. Sci. USA, 99, 3764-3769. 

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