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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 interaction between the ttPOLXc and ttPHP domains. (A) Proteins were analyzed by 7.5% native PAGE and stained with CBB. The samples electrophoresed in the lanes were as follows: 1, 50 pmol ttPolX; 2, 50 pmol ttPOLXc domain; 3, 50 pmol ttPHP domain; and 4, 50 pmol ttPOLXc domain and 50 pmol ttPHP domain. The positions of the proteins on the gel are indicated on right side of the figure. (B) Elution profiles of ttPolX (black line), the ttPOLXc domain (light gray line), the ttPHP domain (dark gray line) and the domain mixture (dotted black line) on size exclusion chromatography. Proteins (500 pmol each) were incubated in buffer comprising 20 mM Tris–HCl and 100 mM KCl with or without 10 μM dNTPs, pH 8.0, at 37°C. The mixtures were incubated for 30 min at 37°C and then applied to a Superdex 75 column and eluted with incubation buffer. The elution volume, absorbance values and MWs estimated from the elution volume of each peak are summarized in Table 1. Arrows in the graph indicate the peaks in each elution profile. In the profile of the domain mixture, there were two main peaks. We named their peaks 1 and 2 in elution order. (C) Elution profiles of the domain mixture in the presence (dotted light gray line) and absence (dotted black line) of 10 μM dNTPs. The eluted peaks were named as described above.
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Figure 7: The interaction between the ttPOLXc and ttPHP domains. (A) Proteins were analyzed by 7.5% native PAGE and stained with CBB. The samples electrophoresed in the lanes were as follows: 1, 50 pmol ttPolX; 2, 50 pmol ttPOLXc domain; 3, 50 pmol ttPHP domain; and 4, 50 pmol ttPOLXc domain and 50 pmol ttPHP domain. The positions of the proteins on the gel are indicated on right side of the figure. (B) Elution profiles of ttPolX (black line), the ttPOLXc domain (light gray line), the ttPHP domain (dark gray line) and the domain mixture (dotted black line) on size exclusion chromatography. Proteins (500 pmol each) were incubated in buffer comprising 20 mM Tris–HCl and 100 mM KCl with or without 10 μM dNTPs, pH 8.0, at 37°C. The mixtures were incubated for 30 min at 37°C and then applied to a Superdex 75 column and eluted with incubation buffer. The elution volume, absorbance values and MWs estimated from the elution volume of each peak are summarized in Table 1. Arrows in the graph indicate the peaks in each elution profile. In the profile of the domain mixture, there were two main peaks. We named their peaks 1 and 2 in elution order. (C) Elution profiles of the domain mixture in the presence (dotted light gray line) and absence (dotted black line) of 10 μM dNTPs. The eluted peaks were named as described above.

Mentions: To verify whether the ttPOLXc and ttPHP domains interact with each other, we performed native PAGE. On the polyacrylamide gel, the ttPolX, ttPOLXc and ttPHP domains showed mobility corresponding to their pI values (Figure 7A): 6.26, 8.40 and 6.04, respectively. It was confirmed by PMF analysis that all bands in the lane of the ttPHP domain contained the ttPHP domain (Figure 7A, lane 3). In the lane of the domain mixture, the bands with the same mobility as ttPolX were apparently increased compared with the lanes of the ttPOLXc and ttPHP domains (Figure 7A). This mobility shift suggests interaction between the ttPOLXc and ttPHP domains.Figure 7.


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 interaction between the ttPOLXc and ttPHP domains. (A) Proteins were analyzed by 7.5% native PAGE and stained with CBB. The samples electrophoresed in the lanes were as follows: 1, 50 pmol ttPolX; 2, 50 pmol ttPOLXc domain; 3, 50 pmol ttPHP domain; and 4, 50 pmol ttPOLXc domain and 50 pmol ttPHP domain. The positions of the proteins on the gel are indicated on right side of the figure. (B) Elution profiles of ttPolX (black line), the ttPOLXc domain (light gray line), the ttPHP domain (dark gray line) and the domain mixture (dotted black line) on size exclusion chromatography. Proteins (500 pmol each) were incubated in buffer comprising 20 mM Tris–HCl and 100 mM KCl with or without 10 μM dNTPs, pH 8.0, at 37°C. The mixtures were incubated for 30 min at 37°C and then applied to a Superdex 75 column and eluted with incubation buffer. The elution volume, absorbance values and MWs estimated from the elution volume of each peak are summarized in Table 1. Arrows in the graph indicate the peaks in each elution profile. In the profile of the domain mixture, there were two main peaks. We named their peaks 1 and 2 in elution order. (C) Elution profiles of the domain mixture in the presence (dotted light gray line) and absence (dotted black line) of 10 μM dNTPs. The eluted peaks were named as described above.
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Figure 7: The interaction between the ttPOLXc and ttPHP domains. (A) Proteins were analyzed by 7.5% native PAGE and stained with CBB. The samples electrophoresed in the lanes were as follows: 1, 50 pmol ttPolX; 2, 50 pmol ttPOLXc domain; 3, 50 pmol ttPHP domain; and 4, 50 pmol ttPOLXc domain and 50 pmol ttPHP domain. The positions of the proteins on the gel are indicated on right side of the figure. (B) Elution profiles of ttPolX (black line), the ttPOLXc domain (light gray line), the ttPHP domain (dark gray line) and the domain mixture (dotted black line) on size exclusion chromatography. Proteins (500 pmol each) were incubated in buffer comprising 20 mM Tris–HCl and 100 mM KCl with or without 10 μM dNTPs, pH 8.0, at 37°C. The mixtures were incubated for 30 min at 37°C and then applied to a Superdex 75 column and eluted with incubation buffer. The elution volume, absorbance values and MWs estimated from the elution volume of each peak are summarized in Table 1. Arrows in the graph indicate the peaks in each elution profile. In the profile of the domain mixture, there were two main peaks. We named their peaks 1 and 2 in elution order. (C) Elution profiles of the domain mixture in the presence (dotted light gray line) and absence (dotted black line) of 10 μM dNTPs. The eluted peaks were named as described above.
Mentions: To verify whether the ttPOLXc and ttPHP domains interact with each other, we performed native PAGE. On the polyacrylamide gel, the ttPolX, ttPOLXc and ttPHP domains showed mobility corresponding to their pI values (Figure 7A): 6.26, 8.40 and 6.04, respectively. It was confirmed by PMF analysis that all bands in the lane of the ttPHP domain contained the ttPHP domain (Figure 7A, lane 3). In the lane of the domain mixture, the bands with the same mobility as ttPolX were apparently increased compared with the lanes of the ttPOLXc and ttPHP domains (Figure 7A). This mobility shift suggests interaction between the ttPOLXc and ttPHP domains.Figure 7.

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