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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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Purified ttPolX and its domains. The purified proteins were analyzed by 12.5% SDS–PAGE and stained with Coomassie Brilliant Blue (CBB). M represents molecular weight markers. The theoretical molecular weights of ttPolX and the His-tagged domains were 64, 44 and 29 kDa, respectively.
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Figure 2: Purified ttPolX and its domains. The purified proteins were analyzed by 12.5% SDS–PAGE and stained with Coomassie Brilliant Blue (CBB). M represents molecular weight markers. The theoretical molecular weights of ttPolX and the His-tagged domains were 64, 44 and 29 kDa, respectively.

Mentions: The sequence of ttha1150 encodes ttPolX of 575 amino acid residues. ttPolX has a calculated molecular mass of 64 kDa with a theoretical isoelectric point of 6.3. We overexpressed ttPolX in E. coli and purified the protein to homogeneity by means of heat treatment and four column chromatography steps (Figure 2). Approximately 20 mg of ttPolX was obtained from 10 g of cells.Figure 2.


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)

Purified ttPolX and its domains. The purified proteins were analyzed by 12.5% SDS–PAGE and stained with Coomassie Brilliant Blue (CBB). M represents molecular weight markers. The theoretical molecular weights of ttPolX and the His-tagged domains were 64, 44 and 29 kDa, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665239&req=5

Figure 2: Purified ttPolX and its domains. The purified proteins were analyzed by 12.5% SDS–PAGE and stained with Coomassie Brilliant Blue (CBB). M represents molecular weight markers. The theoretical molecular weights of ttPolX and the His-tagged domains were 64, 44 and 29 kDa, respectively.
Mentions: The sequence of ttha1150 encodes ttPolX of 575 amino acid residues. ttPolX has a calculated molecular mass of 64 kDa with a theoretical isoelectric point of 6.3. We overexpressed ttPolX in E. coli and purified the protein to homogeneity by means of heat treatment and four column chromatography steps (Figure 2). Approximately 20 mg of ttPolX was obtained from 10 g of cells.Figure 2.

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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