In vivo protein interactions within the Escherichia coli DNA polymerase III core

Autor: Zygmunt Cieśla, Adrianna Nowicka, Iwona J. Fijalkowska, Piotr Jonczyk, Roel M. Schaaper
Rok vydání: 1998
Předmět:
Zdroj: Journal of bacteriology. 180(6)
ISSN: 0021-9193
Popis: Replication of the Escherichia coli chromosome is performed by the DNA polymerase III holoenzyme (Pol III HE) (15, 16, 21). Pol III HE is an asymmetric, dimeric complex containing a total of 18 subunits (10 distinct), which are capable of coordinately synthesizing leading and lagging strands. The complex contains two polymerase cores, one for each strand, composed of α, ɛ, and θ subunits (22). With regard to the fidelity of the replication process, the α and ɛ subunits of the Pol III core are of particular importance. The α subunit (dnaE gene product) is the DNA polymerase, which selects the correct nucleotides during template-directed DNA synthesis (18). The ɛ subunit (dnaQ gene product) performs the 3′→5′ exonucleolytic proofreading activity, which preferentially removes incorrect bases inserted by the polymerase (27). The function of the third subunit, the θ subunit, has yet to be identified. The ɛ subunit of the Pol III HE plays a complex role in DNA replication. Besides its proofreading activity, it stabilizes the core by tightly binding to both the α and θ subunits (22, 29). Interestingly, the α and ɛ subunits are each less active individually than when bound together in the Pol III core (19). One may hypothesize that the fidelity of DNA replication depends not only on the intrinsic accuracy of the polymerase and the strength of the 3′→5′ exonuclease activity but also on the appropriate interactions between the subunits within the Pol III core. Thus, the decreased fidelity of DNA replication observed in E. coli strains carrying mutations within the dnaQ gene could be due either to the defective catalytic properties of the ɛ subunit or to aberrant subunit interactions within the Pol III core. Several mutators which carry mutations in the dnaQ gene have been isolated, e.g., dnaQ49, mutD5, and dnaQ926 strains. Two of these mutations, mutD5 and dnaQ49, have been extensively studied. mutD5 is a particularly strong mutator allele, leading to mutation rates of up to 105-fold above the wild-type level (3, 4). This is due not only to the proofreading defect but also to the concomitant impairment (by saturation) of postreplicative mismatch repair (25, 26). dnaQ49 strains differ from mutD5 strains in several respects. First, dnaQ49 is a temperature-sensitive mutator, possessing modest mutator activity below 30°C but strongly enhanced activity at 37°C (7, 10, 11). Second, dnaQ49 strains are unable to grow at 44.5°C in salt-free rich medium because of the inhibition of DNA synthesis (10). Third, the dnaQ49 allele is recessive with respect to the wild-type gene, while mutD5 is dominant (3, 20). The dnaQ49 and mutD5 alleles result from different missense mutations within the dnaQ gene (9, 30; see also Table ​Table1).1). On the basis of genetic data, a model in which the DnaQ49 protein has a reduced ability to bind to the α subunit has been proposed (30). The third allele, dnaQ926, is the strongest known mutator of E. coli (9). It was constructed by site-specific mutagenesis by changing the codons for two conserved amino acid residues in the ExoI motif of the ɛ subunit (Table ​(Table1)1) known to be essential for the catalytic activity of other polymerase-associated proofreading exonucleases (1). When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, may still efficiently bind to the α subunit. When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells were essentially inviable. dnaQ926 strains survived well, however, when carrying a dnaE antimutator mutation (6, 8) or a multicopy plasmid containing the E. coli mutL+ gene. Thus, the poor viability of dnaQ926 strains was proposed to result from excessively high mutation rates due, as in the case of mutD5 strains, both to the proofreading defect and to the collapse of the mismatch repair system (error catastrophe). TABLE 1 dnaQ mutants tested in this work A mechanism coordinating DNA polymerization and DNA excision, relying on structural and functional communication between the different subunits of Pol III HE, may play an important role in maintaining optimal fidelity of DNA replication. We have been particularly interested in elucidating the physiological role of interactions between the α and ɛ subunits. In an attempt to achieve this goal, we used the Saccharomyces cerevisiae two-hybrid system to investigate the in vivo interactions between mutant and wild-type DnaQ protein with the wild-type DnaE and HolE proteins.
Databáze: OpenAIRE
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