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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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EMSA of ttPolX, and the ttPOLXc and ttPHP domains against four kinds of DNA. (A) Proteins were mixed with 10 nM each DNA and then analyzed by 5% PAGE. The concentrations of ttPolX were 0, 0.5, 1, 2, 5 and 10 μM. The concentrations of the ttPOLXc domain were 0, 0.625, 1.25, 2.5, 5 and 10 μM. The concentrations of the ttPHP domain were 0, 0.01, 0.0625, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 3 and 6.5 μM. (B) The percentage of the protein–DNA complex was determined and plotted against the protein concentration. The symbols were as follows: circles, ssDNA; squares, dsDNA; triangles, 1-nt gapped DNA; diamonds, 5′-phosphorylated 1-nt gapped DNA. The data were fitted with equation (4) using Igor 4.03. Data represent the means for four or five independent experiments ± standard deviation. (C) Sequence alignment of the POLXc domain regions of PolXs. White letters in black boxes indicate putative residues interacting with 5′-phosphate in the crystal structures of hsPolμ (PDB ID: 2fms), hsPolλ (PDB ID: 1xsn) and mmPolμ (PDB ID: 2ihm). The residues with a gray background are putative residues interacting with 5′-phosphate predicted from the alignment. B1, B2 and B3 represent the positions of the basic amino acid residues. The alignment was performed with ClustalW2 (28). The accession numbers were as follows: NP_002681 for H. sapiens Polβ (hsPolβ); NP_037406 for H. sapiens Polλ (hsPolλ); NP_059097 for Mus musculus Polμ (mmPolμ); NP_009940 for Saccharomyces cerevisiae PolIV (scPolIV); NP_592977 for Schizosaccharomyces pombe PolIV (spPolIV); and YP_144416 for T. thermophilus HB8 PolX (ttPolX).
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Figure 9: EMSA of ttPolX, and the ttPOLXc and ttPHP domains against four kinds of DNA. (A) Proteins were mixed with 10 nM each DNA and then analyzed by 5% PAGE. The concentrations of ttPolX were 0, 0.5, 1, 2, 5 and 10 μM. The concentrations of the ttPOLXc domain were 0, 0.625, 1.25, 2.5, 5 and 10 μM. The concentrations of the ttPHP domain were 0, 0.01, 0.0625, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 3 and 6.5 μM. (B) The percentage of the protein–DNA complex was determined and plotted against the protein concentration. The symbols were as follows: circles, ssDNA; squares, dsDNA; triangles, 1-nt gapped DNA; diamonds, 5′-phosphorylated 1-nt gapped DNA. The data were fitted with equation (4) using Igor 4.03. Data represent the means for four or five independent experiments ± standard deviation. (C) Sequence alignment of the POLXc domain regions of PolXs. White letters in black boxes indicate putative residues interacting with 5′-phosphate in the crystal structures of hsPolμ (PDB ID: 2fms), hsPolλ (PDB ID: 1xsn) and mmPolμ (PDB ID: 2ihm). The residues with a gray background are putative residues interacting with 5′-phosphate predicted from the alignment. B1, B2 and B3 represent the positions of the basic amino acid residues. The alignment was performed with ClustalW2 (28). The accession numbers were as follows: NP_002681 for H. sapiens Polβ (hsPolβ); NP_037406 for H. sapiens Polλ (hsPolλ); NP_059097 for Mus musculus Polμ (mmPolμ); NP_009940 for Saccharomyces cerevisiae PolIV (scPolIV); NP_592977 for Schizosaccharomyces pombe PolIV (spPolIV); and YP_144416 for T. thermophilus HB8 PolX (ttPolX).

Mentions: Finally, we examined the DNA-binding abilities of ttPolX and its domains by means of EMSA. We used four DNAs of different structures: ssDNA, dsDNA, 1-nt gapped DNA and 5′-phosphorylated 1-nt gapped DNA. ttPolX and the ttPOLXc domain were able to bind to all DNA substrates but exhibited different binding properties for the four DNA structures (Figure 9A and B, and Table 2). ttPolX showed stronger binding ability as to gapped DNAs than ssDNA and dsDNA. The ttPOLXc domain showed weaker binding ability as to all DNA substrates than ttPolX (Figure 9A and B). Additionally, the ttPOLXc domain showed no significant difference in binding ability between gapped DNA and nongapped DNA. It should be mentioned that the binding curves were sigmoidal (Figure 9B). Assuming multiple molecules of a protein bind to DNA, we determined Kdapp and n (the number of bound protein) (see Materials and methods section). The ttPHP domain was unable to bind to all of the DNA structures, even though the concentration of ttPHP domain was 6.5 μM (Figure 9A and 9B). We observed more than two shifted bands in some lanes for ttPolX and the ttPOLXc domain. The appearance of multiple shifted bands and the n values suggest that more than two molecules of ttPolX or the ttPOLXc domain bound to a DNA. There was almost no difference between the binding abilities as to 5′-phosphorylated and unphosphorylated 1-nt gapped DNA (Figure 9A and B, and Table 2). Figure 9C shows that putative residues which recognize 5′-phosphate are conserved in various PolXs (see Discussion section).Figure 9.


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)

EMSA of ttPolX, and the ttPOLXc and ttPHP domains against four kinds of DNA. (A) Proteins were mixed with 10 nM each DNA and then analyzed by 5% PAGE. The concentrations of ttPolX were 0, 0.5, 1, 2, 5 and 10 μM. The concentrations of the ttPOLXc domain were 0, 0.625, 1.25, 2.5, 5 and 10 μM. The concentrations of the ttPHP domain were 0, 0.01, 0.0625, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 3 and 6.5 μM. (B) The percentage of the protein–DNA complex was determined and plotted against the protein concentration. The symbols were as follows: circles, ssDNA; squares, dsDNA; triangles, 1-nt gapped DNA; diamonds, 5′-phosphorylated 1-nt gapped DNA. The data were fitted with equation (4) using Igor 4.03. Data represent the means for four or five independent experiments ± standard deviation. (C) Sequence alignment of the POLXc domain regions of PolXs. White letters in black boxes indicate putative residues interacting with 5′-phosphate in the crystal structures of hsPolμ (PDB ID: 2fms), hsPolλ (PDB ID: 1xsn) and mmPolμ (PDB ID: 2ihm). The residues with a gray background are putative residues interacting with 5′-phosphate predicted from the alignment. B1, B2 and B3 represent the positions of the basic amino acid residues. The alignment was performed with ClustalW2 (28). The accession numbers were as follows: NP_002681 for H. sapiens Polβ (hsPolβ); NP_037406 for H. sapiens Polλ (hsPolλ); NP_059097 for Mus musculus Polμ (mmPolμ); NP_009940 for Saccharomyces cerevisiae PolIV (scPolIV); NP_592977 for Schizosaccharomyces pombe PolIV (spPolIV); and YP_144416 for T. thermophilus HB8 PolX (ttPolX).
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Figure 9: EMSA of ttPolX, and the ttPOLXc and ttPHP domains against four kinds of DNA. (A) Proteins were mixed with 10 nM each DNA and then analyzed by 5% PAGE. The concentrations of ttPolX were 0, 0.5, 1, 2, 5 and 10 μM. The concentrations of the ttPOLXc domain were 0, 0.625, 1.25, 2.5, 5 and 10 μM. The concentrations of the ttPHP domain were 0, 0.01, 0.0625, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 3 and 6.5 μM. (B) The percentage of the protein–DNA complex was determined and plotted against the protein concentration. The symbols were as follows: circles, ssDNA; squares, dsDNA; triangles, 1-nt gapped DNA; diamonds, 5′-phosphorylated 1-nt gapped DNA. The data were fitted with equation (4) using Igor 4.03. Data represent the means for four or five independent experiments ± standard deviation. (C) Sequence alignment of the POLXc domain regions of PolXs. White letters in black boxes indicate putative residues interacting with 5′-phosphate in the crystal structures of hsPolμ (PDB ID: 2fms), hsPolλ (PDB ID: 1xsn) and mmPolμ (PDB ID: 2ihm). The residues with a gray background are putative residues interacting with 5′-phosphate predicted from the alignment. B1, B2 and B3 represent the positions of the basic amino acid residues. The alignment was performed with ClustalW2 (28). The accession numbers were as follows: NP_002681 for H. sapiens Polβ (hsPolβ); NP_037406 for H. sapiens Polλ (hsPolλ); NP_059097 for Mus musculus Polμ (mmPolμ); NP_009940 for Saccharomyces cerevisiae PolIV (scPolIV); NP_592977 for Schizosaccharomyces pombe PolIV (spPolIV); and YP_144416 for T. thermophilus HB8 PolX (ttPolX).
Mentions: Finally, we examined the DNA-binding abilities of ttPolX and its domains by means of EMSA. We used four DNAs of different structures: ssDNA, dsDNA, 1-nt gapped DNA and 5′-phosphorylated 1-nt gapped DNA. ttPolX and the ttPOLXc domain were able to bind to all DNA substrates but exhibited different binding properties for the four DNA structures (Figure 9A and B, and Table 2). ttPolX showed stronger binding ability as to gapped DNAs than ssDNA and dsDNA. The ttPOLXc domain showed weaker binding ability as to all DNA substrates than ttPolX (Figure 9A and B). Additionally, the ttPOLXc domain showed no significant difference in binding ability between gapped DNA and nongapped DNA. It should be mentioned that the binding curves were sigmoidal (Figure 9B). Assuming multiple molecules of a protein bind to DNA, we determined Kdapp and n (the number of bound protein) (see Materials and methods section). The ttPHP domain was unable to bind to all of the DNA structures, even though the concentration of ttPHP domain was 6.5 μM (Figure 9A and 9B). We observed more than two shifted bands in some lanes for ttPolX and the ttPOLXc domain. The appearance of multiple shifted bands and the n values suggest that more than two molecules of ttPolX or the ttPOLXc domain bound to a DNA. There was almost no difference between the binding abilities as to 5′-phosphorylated and unphosphorylated 1-nt gapped DNA (Figure 9A and B, and Table 2). Figure 9C shows that putative residues which recognize 5′-phosphate are conserved in various PolXs (see Discussion section).Figure 9.

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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