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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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Limited proteolysis of Pol θ with trypsin. (A) Purified 98-kDa Pol θ (66 µg) was digested at 25°C with trypsin (0.66 µg) at a weight ratio (trypsin:Pol θ) of 1:100 in 100 mM Tris–HCl, pH 8.0. Aliquots were withdrawn at 5-, 15-, 30- and 60-min time intervals, as indicated at the ‘top’ of the photograph. A portion of each digested sample was mixed with 10 µl SDS–PAGE gel-loading buffer and resolved on a 12% NuPAGE Bis–Tris gel. The proteins were transferred onto a PVDF membrane for amino-terminal sequencing, as described under Materials and Methods section. The positions of Pol θ, tryptic peptides and protein markers are indicated. (B) Amino-terminal sequencing was performed using the Procise sequencing system, Model 492 (Applied Biosystems). The amino-terminal sequences of peptides I–VI are shown. (C) The domain organization of 98-kDa Pol θ is depicted. (D) Portions of the trypsin digested samples from (A) were reacted with 5′-end-labeled dRP lyase DNA substrate and subjected to NaBH4 cross-linking as in Figure 5. The gel was scanned on a PhosphorImager. The positions of cross-linked Pol θ, 23- and 24-kDa peptides are indicated. (E) The gel in (D) was stained with silver to detect protein fragments. The positions of Pol θ, 23- and 24-kDA peptides are indicated.
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Figure 6: Limited proteolysis of Pol θ with trypsin. (A) Purified 98-kDa Pol θ (66 µg) was digested at 25°C with trypsin (0.66 µg) at a weight ratio (trypsin:Pol θ) of 1:100 in 100 mM Tris–HCl, pH 8.0. Aliquots were withdrawn at 5-, 15-, 30- and 60-min time intervals, as indicated at the ‘top’ of the photograph. A portion of each digested sample was mixed with 10 µl SDS–PAGE gel-loading buffer and resolved on a 12% NuPAGE Bis–Tris gel. The proteins were transferred onto a PVDF membrane for amino-terminal sequencing, as described under Materials and Methods section. The positions of Pol θ, tryptic peptides and protein markers are indicated. (B) Amino-terminal sequencing was performed using the Procise sequencing system, Model 492 (Applied Biosystems). The amino-terminal sequences of peptides I–VI are shown. (C) The domain organization of 98-kDa Pol θ is depicted. (D) Portions of the trypsin digested samples from (A) were reacted with 5′-end-labeled dRP lyase DNA substrate and subjected to NaBH4 cross-linking as in Figure 5. The gel was scanned on a PhosphorImager. The positions of cross-linked Pol θ, 23- and 24-kDa peptides are indicated. (E) The gel in (D) was stained with silver to detect protein fragments. The positions of Pol θ, 23- and 24-kDA peptides are indicated.

Mentions: To probe domain organization of the Pol θ 98-kDa polymerase domain and to localize the 5′-dRP lyase active site of Pol θ, we subjected the purified protein to controlled proteolysis with trypsin, and then we identified the digestion products by amino-terminal sequencing. Samples from the incubation with trypsin were withdrawn at different time intervals. A portion of each sample was separated by SDS–PAGE, transferred onto a PVDF membrane and subjected to amino-terminal sequencing as described under ‘Materials and Methods’ section (Figure 6). Digestion for 5 min completely degraded the 98-kDa enzyme and produced two major fragments with molecular masses of about 33 and 24 kDa; several minor polypeptides of higher and lower molecular masses also were produced. With longer incubations of 30 and 60 min, four fragments persisted and were designated as fragments II, III, IV and V, respectively (Figure 6A). The 24-kDa fragment appeared to be further digested to a smaller molecular mass polypeptide resulting in a doublet in the gel, whereas the 20-kDa polypeptide and some high-molecular-mass polypeptides almost disappeared (Figure 6A). Amino-terminal sequence analysis of several of these protease-resistant fragments (denoted as I–VI in Figure 6A) revealed that the 33-, 24-, 23-, 14- and 9-kDA polypeptides (II to VI) begin with Phe124, Cys545, Gly557, Leu423 and Asn41, respectively (Figure 6B). These results indicated that the 23-kDa (IV) polypeptide resulted from removal of 12 amino acids from the amino terminus of the 24-kDa (III) polypeptide. Fragment I, with a molecular mass of 42 kDa, failed to exhibit reasonable sequence data due to low abundance. The domains of the 98-kDa Pol θ were aligned (Figure 6C) based on the molecular masses of these protease-resistant fragments and their amino-terminal sequences.Figure 6.


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)

Limited proteolysis of Pol θ with trypsin. (A) Purified 98-kDa Pol θ (66 µg) was digested at 25°C with trypsin (0.66 µg) at a weight ratio (trypsin:Pol θ) of 1:100 in 100 mM Tris–HCl, pH 8.0. Aliquots were withdrawn at 5-, 15-, 30- and 60-min time intervals, as indicated at the ‘top’ of the photograph. A portion of each digested sample was mixed with 10 µl SDS–PAGE gel-loading buffer and resolved on a 12% NuPAGE Bis–Tris gel. The proteins were transferred onto a PVDF membrane for amino-terminal sequencing, as described under Materials and Methods section. The positions of Pol θ, tryptic peptides and protein markers are indicated. (B) Amino-terminal sequencing was performed using the Procise sequencing system, Model 492 (Applied Biosystems). The amino-terminal sequences of peptides I–VI are shown. (C) The domain organization of 98-kDa Pol θ is depicted. (D) Portions of the trypsin digested samples from (A) were reacted with 5′-end-labeled dRP lyase DNA substrate and subjected to NaBH4 cross-linking as in Figure 5. The gel was scanned on a PhosphorImager. The positions of cross-linked Pol θ, 23- and 24-kDa peptides are indicated. (E) The gel in (D) was stained with silver to detect protein fragments. The positions of Pol θ, 23- and 24-kDA peptides are indicated.
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Figure 6: Limited proteolysis of Pol θ with trypsin. (A) Purified 98-kDa Pol θ (66 µg) was digested at 25°C with trypsin (0.66 µg) at a weight ratio (trypsin:Pol θ) of 1:100 in 100 mM Tris–HCl, pH 8.0. Aliquots were withdrawn at 5-, 15-, 30- and 60-min time intervals, as indicated at the ‘top’ of the photograph. A portion of each digested sample was mixed with 10 µl SDS–PAGE gel-loading buffer and resolved on a 12% NuPAGE Bis–Tris gel. The proteins were transferred onto a PVDF membrane for amino-terminal sequencing, as described under Materials and Methods section. The positions of Pol θ, tryptic peptides and protein markers are indicated. (B) Amino-terminal sequencing was performed using the Procise sequencing system, Model 492 (Applied Biosystems). The amino-terminal sequences of peptides I–VI are shown. (C) The domain organization of 98-kDa Pol θ is depicted. (D) Portions of the trypsin digested samples from (A) were reacted with 5′-end-labeled dRP lyase DNA substrate and subjected to NaBH4 cross-linking as in Figure 5. The gel was scanned on a PhosphorImager. The positions of cross-linked Pol θ, 23- and 24-kDa peptides are indicated. (E) The gel in (D) was stained with silver to detect protein fragments. The positions of Pol θ, 23- and 24-kDA peptides are indicated.
Mentions: To probe domain organization of the Pol θ 98-kDa polymerase domain and to localize the 5′-dRP lyase active site of Pol θ, we subjected the purified protein to controlled proteolysis with trypsin, and then we identified the digestion products by amino-terminal sequencing. Samples from the incubation with trypsin were withdrawn at different time intervals. A portion of each sample was separated by SDS–PAGE, transferred onto a PVDF membrane and subjected to amino-terminal sequencing as described under ‘Materials and Methods’ section (Figure 6). Digestion for 5 min completely degraded the 98-kDa enzyme and produced two major fragments with molecular masses of about 33 and 24 kDa; several minor polypeptides of higher and lower molecular masses also were produced. With longer incubations of 30 and 60 min, four fragments persisted and were designated as fragments II, III, IV and V, respectively (Figure 6A). The 24-kDa fragment appeared to be further digested to a smaller molecular mass polypeptide resulting in a doublet in the gel, whereas the 20-kDa polypeptide and some high-molecular-mass polypeptides almost disappeared (Figure 6A). Amino-terminal sequence analysis of several of these protease-resistant fragments (denoted as I–VI in Figure 6A) revealed that the 33-, 24-, 23-, 14- and 9-kDA polypeptides (II to VI) begin with Phe124, Cys545, Gly557, Leu423 and Asn41, respectively (Figure 6B). These results indicated that the 23-kDa (IV) polypeptide resulted from removal of 12 amino acids from the amino terminus of the 24-kDa (III) polypeptide. Fragment I, with a molecular mass of 42 kDa, failed to exhibit reasonable sequence data due to low abundance. The domains of the 98-kDa Pol θ were aligned (Figure 6C) based on the molecular masses of these protease-resistant fragments and their amino-terminal sequences.Figure 6.

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