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Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

Nakane S, Nakagawa N, Kuramitsu S, Masui R - Nucleic Acids Res. (2009)

Bottom Line: The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms.We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities.Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.

ABSTRACT
The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

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The activities of the mixture of the ttPOLXc and ttPHP domains. The measurement conditions were the same as given in Figure 4 except for the use of domain mixture (containing 1 μM ttPOLXc and 1 μM ttPHP domains). (A) Metal ion dependence of polymerase activity. (B) Time course analysis of exonuclease activity against ssDNA and dsDNA. (C) Exonuclease assay with dNTPs. Reaction mixtures comprised 20 mM Tris–HCl, 20 mM KCl, 1 mM MnCl2, 100 nM ttPolX or 1 μM domain mixture, 10 nM 5′-labeled 21F and indicated amount of dNTPs, pH 8.0, at 37°C. The reaction mixtures were incubated for 30 min at 37°C, and then analyzed by 20% denaturing PAGE (8 M urea), but the running time was longer than that in (B). Note that the concentration of the domain mixture was higher than that of ttPolX.
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Figure 6: The activities of the mixture of the ttPOLXc and ttPHP domains. The measurement conditions were the same as given in Figure 4 except for the use of domain mixture (containing 1 μM ttPOLXc and 1 μM ttPHP domains). (A) Metal ion dependence of polymerase activity. (B) Time course analysis of exonuclease activity against ssDNA and dsDNA. (C) Exonuclease assay with dNTPs. Reaction mixtures comprised 20 mM Tris–HCl, 20 mM KCl, 1 mM MnCl2, 100 nM ttPolX or 1 μM domain mixture, 10 nM 5′-labeled 21F and indicated amount of dNTPs, pH 8.0, at 37°C. The reaction mixtures were incubated for 30 min at 37°C, and then analyzed by 20% denaturing PAGE (8 M urea), but the running time was longer than that in (B). Note that the concentration of the domain mixture was higher than that of ttPolX.

Mentions: A mixture of the ttPOLXc and ttPHP domains (domain mixture) had Mn2+-dependent 3′–5′ exonuclease activity as well as Mg2+-dependent DNA polymerase activity (Figure 6A). This metal ion selectivity of the activities was the same as that of ttPolX. These results suggest interaction between the ttPOLXc and ttPHP domains. Time course analysis of the exonuclease activity showed that the domain mixture was able to degrade both ssDNA and dsDNA but the activity for ssDNA was higher than that for dsDNA (Figure 6B). Furthermore, we found that the exonuclease activity of the domain mixture increased in the presence of 0.1–10 μM dNTPs, whereas that of ttPolX did not change with dNTPs (Figure 6C). We calculated steady-state kinetic parameters for the 3′–5′ exonuclease activity of ttPolX and the domain mixture against 3′-labeled 21-mer ssDNA (see Materials and methods section). The Km values of ttPolX were 1700 (without dNTPs) and 1300 nM (with dNTPs), whereas those of the domain mixture were 770 (without dNTPs) and 530 nM (with dNTPs). The catalytic efficiencies (kcat/Km) of ttPolX were 2.8 × 104 (without dNTPs) and 3.8 × 104 s−1M−1 (with dNTPs), whereas those of the domain mixture were 3.5 × 102 (without dNTPs) and 1.5 × 103 s−1M−1 (with dNTPs). The catalytic efficiency of ttPolX was almost the same with and without dNTPs, whereas that of the domain mixture increased on the addition of dNTPs.Figure 6.


Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

Nakane S, Nakagawa N, Kuramitsu S, Masui R - Nucleic Acids Res. (2009)

The activities of the mixture of the ttPOLXc and ttPHP domains. The measurement conditions were the same as given in Figure 4 except for the use of domain mixture (containing 1 μM ttPOLXc and 1 μM ttPHP domains). (A) Metal ion dependence of polymerase activity. (B) Time course analysis of exonuclease activity against ssDNA and dsDNA. (C) Exonuclease assay with dNTPs. Reaction mixtures comprised 20 mM Tris–HCl, 20 mM KCl, 1 mM MnCl2, 100 nM ttPolX or 1 μM domain mixture, 10 nM 5′-labeled 21F and indicated amount of dNTPs, pH 8.0, at 37°C. The reaction mixtures were incubated for 30 min at 37°C, and then analyzed by 20% denaturing PAGE (8 M urea), but the running time was longer than that in (B). Note that the concentration of the domain mixture was higher than that of ttPolX.
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Figure 6: The activities of the mixture of the ttPOLXc and ttPHP domains. The measurement conditions were the same as given in Figure 4 except for the use of domain mixture (containing 1 μM ttPOLXc and 1 μM ttPHP domains). (A) Metal ion dependence of polymerase activity. (B) Time course analysis of exonuclease activity against ssDNA and dsDNA. (C) Exonuclease assay with dNTPs. Reaction mixtures comprised 20 mM Tris–HCl, 20 mM KCl, 1 mM MnCl2, 100 nM ttPolX or 1 μM domain mixture, 10 nM 5′-labeled 21F and indicated amount of dNTPs, pH 8.0, at 37°C. The reaction mixtures were incubated for 30 min at 37°C, and then analyzed by 20% denaturing PAGE (8 M urea), but the running time was longer than that in (B). Note that the concentration of the domain mixture was higher than that of ttPolX.
Mentions: A mixture of the ttPOLXc and ttPHP domains (domain mixture) had Mn2+-dependent 3′–5′ exonuclease activity as well as Mg2+-dependent DNA polymerase activity (Figure 6A). This metal ion selectivity of the activities was the same as that of ttPolX. These results suggest interaction between the ttPOLXc and ttPHP domains. Time course analysis of the exonuclease activity showed that the domain mixture was able to degrade both ssDNA and dsDNA but the activity for ssDNA was higher than that for dsDNA (Figure 6B). Furthermore, we found that the exonuclease activity of the domain mixture increased in the presence of 0.1–10 μM dNTPs, whereas that of ttPolX did not change with dNTPs (Figure 6C). We calculated steady-state kinetic parameters for the 3′–5′ exonuclease activity of ttPolX and the domain mixture against 3′-labeled 21-mer ssDNA (see Materials and methods section). The Km values of ttPolX were 1700 (without dNTPs) and 1300 nM (with dNTPs), whereas those of the domain mixture were 770 (without dNTPs) and 530 nM (with dNTPs). The catalytic efficiencies (kcat/Km) of ttPolX were 2.8 × 104 (without dNTPs) and 3.8 × 104 s−1M−1 (with dNTPs), whereas those of the domain mixture were 3.5 × 102 (without dNTPs) and 1.5 × 103 s−1M−1 (with dNTPs). The catalytic efficiency of ttPolX was almost the same with and without dNTPs, whereas that of the domain mixture increased on the addition of dNTPs.Figure 6.

Bottom Line: The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms.We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities.Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.

ABSTRACT
The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

Show MeSH
Related in: MedlinePlus