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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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Domain structures predicted by sequence motif analysis of representative PolXs. The numbers represent the amino acid residues. hsPolλ hsPolμ, hsTdT and hsPolβ represent the enzymes of Homo sapiens. The accession numbers are as follows: NP_037406 for hsPolλ; NP_037416 for hsPolμ; NP_004079 for hsTdT; NP_002681 for hsPolβ and YP_144416 for ttPolX.
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Figure 1: Domain structures predicted by sequence motif analysis of representative PolXs. The numbers represent the amino acid residues. hsPolλ hsPolμ, hsTdT and hsPolβ represent the enzymes of Homo sapiens. The accession numbers are as follows: NP_037406 for hsPolλ; NP_037416 for hsPolμ; NP_004079 for hsTdT; NP_002681 for hsPolβ and YP_144416 for ttPolX.

Mentions: Eukaryotic PolXs are composed of mainly two domains, a PolX core (POLXc) domain at the C-terminus and a BRCA1 (breast cancer susceptibility protein) C-terminal (BRCT) domain at the N-terminus (Figure 1). However, Polβ consists of only a POLXc domain, and Polσs have no POLXc or BRCT domain (14). The POLXc domain possesses polymerase activity and contains two helix-hairpin-helix motifs, which are responsible for sequence-nonspecific DNA binding (15). On the other hand, the BRCT domain interacts with other proteins and/or DNA and joins the Ku-XRCC4-DNA ligase IV-DNA complex for DSBR (16,17). Therefore, although all PolXs are single-subunit enzymes, their domain architecture permits them to exhibit multiple biochemical activities.Figure 1.


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)

Domain structures predicted by sequence motif analysis of representative PolXs. The numbers represent the amino acid residues. hsPolλ hsPolμ, hsTdT and hsPolβ represent the enzymes of Homo sapiens. The accession numbers are as follows: NP_037406 for hsPolλ; NP_037416 for hsPolμ; NP_004079 for hsTdT; NP_002681 for hsPolβ and YP_144416 for ttPolX.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Domain structures predicted by sequence motif analysis of representative PolXs. The numbers represent the amino acid residues. hsPolλ hsPolμ, hsTdT and hsPolβ represent the enzymes of Homo sapiens. The accession numbers are as follows: NP_037406 for hsPolλ; NP_037416 for hsPolμ; NP_004079 for hsTdT; NP_002681 for hsPolβ and YP_144416 for ttPolX.
Mentions: Eukaryotic PolXs are composed of mainly two domains, a PolX core (POLXc) domain at the C-terminus and a BRCA1 (breast cancer susceptibility protein) C-terminal (BRCT) domain at the N-terminus (Figure 1). However, Polβ consists of only a POLXc domain, and Polσs have no POLXc or BRCT domain (14). The POLXc domain possesses polymerase activity and contains two helix-hairpin-helix motifs, which are responsible for sequence-nonspecific DNA binding (15). On the other hand, the BRCT domain interacts with other proteins and/or DNA and joins the Ku-XRCC4-DNA ligase IV-DNA complex for DSBR (16,17). Therefore, although all PolXs are single-subunit enzymes, their domain architecture permits them to exhibit multiple biochemical activities.Figure 1.

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