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Thermodynamics of the DNA structural selectivity of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus.

ABSTRACT: Understanding the thermodynamics of substrate selection by DNA polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the large fragments of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologs. Klentaq, however, does not have a functional proofreading site. Examination of the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and blunt-end double-stranded DNA (ds-DNA) show that the binding selectivity pattern is similar when examined across a wide range of salt concentration, but can significantly differ at any individual salt concentration. For both proteins, binding of single-stranded DNA shifts from weakest to tightest binding of the three structures as the salt concentration increases. Both Klenow and Klentaq release two to three more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA. Klenow exhibits significant differences in the Delta C(p) of binding to pt-DNA versus ds-DNA, and a difference in pI for these two complexes, whereas Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two structures differently. Taken together, the data suggest that the two polymerases bind ds-DNA very differently, but that both bind pt-DNA and ss-DNA similarly, despite the absence of a proofreading site in Klentaq.

SUBMITTER: Wowor AJ

PROVIDER: S-EPMC2884233 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Thermodynamics of the DNA structural selectivity of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus.

Wowor Andy J AJ Datta Kausiki K Brown Hiromi S HS Thompson Gregory S GS Ray Sreerupa S Grove Anne A LiCata Vince J VJ

Biophysical journal 20100601 12

Understanding the thermodynamics of substrate selection by DNA polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the large fragments of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologs. Klentaq, however, does not have a functional proofreading site. Examination of the DNA binding thermodynamics of Klenow and ...[more]

PMID: 20550914

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Project description:Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain. Removal of the 5' nuclease domains produces the 'large fragment' domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature T(m) of 37 and 100 degrees C respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5' nuclease domains of each melting separately. The 5' nuclease domain of Taq denatures as a separate peak, 10 degrees C before the Klentaq domain. Melting of the 5' nuclease domain of Pol 1 overlaps with the Klenow fragment. Presence of the 5' nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower T(m) values, but almost identical van't Hoff enthalpy Delta H values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.

Project description:Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.

Dataset Information

Thermodynamics of the DNA structural selectivity of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus.

Publications

Thermodynamics of the DNA structural selectivity of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus.

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