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Human DNA polymerase theta possesses 5'-dRP lyase activity and functions in single-nucleotide base excision repair in vitro.

Prasad R, Longley MJ, Sharief FS, Hou EW, Copeland WC, Wilson SH - Nucleic Acids Res. (2009)

Bottom Line: The 5'-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5'-dRP lyase activity.Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH(4) cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol theta.These results are consistent with a role of Pol theta in BER.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA.

ABSTRACT
DNA polymerase theta (Pol theta) is a low-fidelity DNA polymerase that belongs to the family A polymerases and has been proposed to play a role in somatic hypermutation. Pol theta has the ability to conduct translesion DNA synthesis opposite an AP site or thymine glycol, and it was recently proposed to be involved in base excision repair (BER) of DNA damage. Here, we show that Pol theta has intrinsic 5'-deoxyribose phosphate (5'-dRP) lyase activity that is involved in single-nucleotide base excision DNA repair (SN-BER). Full-length human Pol theta is a approximately 300-kDa polypeptide, but we show here that the 98-kDa C-terminal region of Pol theta possesses both DNA polymerase activity and dRP lyase activity and is sufficient to carry out base excision repair in vitro. The 5'-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH(4) cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol theta. These results are consistent with a role of Pol theta in BER.

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NaBH4 cross-linking of wild-type and mutant Pol θ. The reaction conditions and product analysis were as described under Materials and Methods section. UDG pretreated DNA substrate (200 nM) as described in Figure 3A was mixed with dilution buffer (lane 1), 20 nM Pol β (lane 2), 150 nM wild-type Pol θ (lane 3) or 150 nM mutant Pol θ (lane 4) and 1 mM NaBH4. The reaction mixtures were incubated on ice for 1 h followed by incubation at room temperature for 10 min. Covalently cross-linked DNA–protein products were separated by 10% NuPAGE Bis–Tris gel, and the gel was scanned on a PhosphorImager. The positions of cross-linked products and free DNA are indicated.
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Figure 5: NaBH4 cross-linking of wild-type and mutant Pol θ. The reaction conditions and product analysis were as described under Materials and Methods section. UDG pretreated DNA substrate (200 nM) as described in Figure 3A was mixed with dilution buffer (lane 1), 20 nM Pol β (lane 2), 150 nM wild-type Pol θ (lane 3) or 150 nM mutant Pol θ (lane 4) and 1 mM NaBH4. The reaction mixtures were incubated on ice for 1 h followed by incubation at room temperature for 10 min. Covalently cross-linked DNA–protein products were separated by 10% NuPAGE Bis–Tris gel, and the gel was scanned on a PhosphorImager. The positions of cross-linked products and free DNA are indicated.

Mentions: To confirm that the 5′-dRP lyase activity of Pol θ was intrinsic to the 98-kDa peptide, we utilized the NaBH4 cross-linking technique (25). This cross-linking technique relies on the ability of the C1′-aldehyde group of the 5′-deoxyribose in the substrate to react with a lysine γ-amine of a protein to form a β-elimination intermediate (Schiff base) protein–DNA complex; this Schiff base is recovered as a covalent bond by NaBH4 trapping. We had previously shown that DNA polymerases, such as Pol β, Pol γ and Pol ι, catalyze dRP removal from the BER intermediate via β-elimination, and the Schiff base intermediate between these polymerases and DNA can be trapped by NaBH4 reduction (27). As demonstrated in Figure 5, both wild-type Pol θ and the polymerase-deficient mutant Pol θ could form covalent protein–DNA products with the BER intermediate substrate upon treatment with NaBH4. This indicates that the Pol θ polymerase domain possesses functional 5′-dRP lyase activity.Figure 5.


Human DNA polymerase theta possesses 5'-dRP lyase activity and functions in single-nucleotide base excision repair in vitro.

Prasad R, Longley MJ, Sharief FS, Hou EW, Copeland WC, Wilson SH - Nucleic Acids Res. (2009)

NaBH4 cross-linking of wild-type and mutant Pol θ. The reaction conditions and product analysis were as described under Materials and Methods section. UDG pretreated DNA substrate (200 nM) as described in Figure 3A was mixed with dilution buffer (lane 1), 20 nM Pol β (lane 2), 150 nM wild-type Pol θ (lane 3) or 150 nM mutant Pol θ (lane 4) and 1 mM NaBH4. The reaction mixtures were incubated on ice for 1 h followed by incubation at room temperature for 10 min. Covalently cross-linked DNA–protein products were separated by 10% NuPAGE Bis–Tris gel, and the gel was scanned on a PhosphorImager. The positions of cross-linked products and free DNA are indicated.
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Related In: Results  -  Collection

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Figure 5: NaBH4 cross-linking of wild-type and mutant Pol θ. The reaction conditions and product analysis were as described under Materials and Methods section. UDG pretreated DNA substrate (200 nM) as described in Figure 3A was mixed with dilution buffer (lane 1), 20 nM Pol β (lane 2), 150 nM wild-type Pol θ (lane 3) or 150 nM mutant Pol θ (lane 4) and 1 mM NaBH4. The reaction mixtures were incubated on ice for 1 h followed by incubation at room temperature for 10 min. Covalently cross-linked DNA–protein products were separated by 10% NuPAGE Bis–Tris gel, and the gel was scanned on a PhosphorImager. The positions of cross-linked products and free DNA are indicated.
Mentions: To confirm that the 5′-dRP lyase activity of Pol θ was intrinsic to the 98-kDa peptide, we utilized the NaBH4 cross-linking technique (25). This cross-linking technique relies on the ability of the C1′-aldehyde group of the 5′-deoxyribose in the substrate to react with a lysine γ-amine of a protein to form a β-elimination intermediate (Schiff base) protein–DNA complex; this Schiff base is recovered as a covalent bond by NaBH4 trapping. We had previously shown that DNA polymerases, such as Pol β, Pol γ and Pol ι, catalyze dRP removal from the BER intermediate via β-elimination, and the Schiff base intermediate between these polymerases and DNA can be trapped by NaBH4 reduction (27). As demonstrated in Figure 5, both wild-type Pol θ and the polymerase-deficient mutant Pol θ could form covalent protein–DNA products with the BER intermediate substrate upon treatment with NaBH4. This indicates that the Pol θ polymerase domain possesses functional 5′-dRP lyase activity.Figure 5.

Bottom Line: The 5'-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5'-dRP lyase activity.Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH(4) cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol theta.These results are consistent with a role of Pol theta in BER.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA.

ABSTRACT
DNA polymerase theta (Pol theta) is a low-fidelity DNA polymerase that belongs to the family A polymerases and has been proposed to play a role in somatic hypermutation. Pol theta has the ability to conduct translesion DNA synthesis opposite an AP site or thymine glycol, and it was recently proposed to be involved in base excision repair (BER) of DNA damage. Here, we show that Pol theta has intrinsic 5'-deoxyribose phosphate (5'-dRP) lyase activity that is involved in single-nucleotide base excision DNA repair (SN-BER). Full-length human Pol theta is a approximately 300-kDa polypeptide, but we show here that the 98-kDa C-terminal region of Pol theta possesses both DNA polymerase activity and dRP lyase activity and is sufficient to carry out base excision repair in vitro. The 5'-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH(4) cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol theta. These results are consistent with a role of Pol theta in BER.

Show MeSH
Related in: MedlinePlus