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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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Kinetic measurements of dRP lyase activity of Pol θ. The loss of dRP from the nicked AP-site containing DNA substrate was examined as a function of incubation time, and the rate of dRP removal by Pol θ was determined under single turnover conditions (i.e. enzyme/DNA = 4). The reaction was performed with Pol θ as described under Materials and Methods section. (A) Schematic representation of the DNA substrate prepared by annealing a 15-mer oligonucleotide, a 17-mer oligonucleotide containing 32P-labeled uracil at the 5′-end, and a 34-mer oligonucleotide template. (B) The duplex DNA (100 nM) was pretreated with UDG and then reacted with 400 nM Pol θ. Aliquots were withdrawn at the indicated time intervals and stabilized by the addition of 20 mM NaBH4 and incubation on ice for 30 min. Then, an equal volume of gel-loading dye buffer was added, and the reaction products were separated by precast 15% TBE-Urea gels. A representative phosphorimage of the polyacrylamide gel illustrates product analysis at various times (lanes 1–5) with and without Pol θ. (C) Time course data were analyzed using ImageQuant software and fitted to an exponential equation by using nonlinear least squares methods to determine the reaction rate (kobs= 0.14/min).
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Figure 3: Kinetic measurements of dRP lyase activity of Pol θ. The loss of dRP from the nicked AP-site containing DNA substrate was examined as a function of incubation time, and the rate of dRP removal by Pol θ was determined under single turnover conditions (i.e. enzyme/DNA = 4). The reaction was performed with Pol θ as described under Materials and Methods section. (A) Schematic representation of the DNA substrate prepared by annealing a 15-mer oligonucleotide, a 17-mer oligonucleotide containing 32P-labeled uracil at the 5′-end, and a 34-mer oligonucleotide template. (B) The duplex DNA (100 nM) was pretreated with UDG and then reacted with 400 nM Pol θ. Aliquots were withdrawn at the indicated time intervals and stabilized by the addition of 20 mM NaBH4 and incubation on ice for 30 min. Then, an equal volume of gel-loading dye buffer was added, and the reaction products were separated by precast 15% TBE-Urea gels. A representative phosphorimage of the polyacrylamide gel illustrates product analysis at various times (lanes 1–5) with and without Pol θ. (C) Time course data were analyzed using ImageQuant software and fitted to an exponential equation by using nonlinear least squares methods to determine the reaction rate (kobs= 0.14/min).

Mentions: To determine kinetic parameters of 5′-dRP group removal by Pol θ, we prepared a 34-bp duplex DNA that contained uracil at position 16 and a nick between positions 15 and 16, by annealing both a 15-mer and a 5′-end 32P-labeled uracil-containing 17-mer to the 34-mer complementary DNA strand. The resulting 32P-labeled nicked DNA was pretreated with UDG to generate the 5′-dRP-containing single-nucleotide gapped substrate; the DNA substrate, thus prepared, contained a 32P-labeled dRP flap in a single-nucleotide gap (Figure 3A, 16-mer + 32P-dRP). The rate of dRP removal by Pol θ was determined under single turnover conditions (i.e. enzyme/DNA = 4). The results revealed that Pol θ cleaved 5′ dRP in a time-dependent manner as monitored by disappearance of the substrate (Figure 3B), with an observed rate of dRP removal of ∼0.14/min (Figure 3C). In other experiments where substrate was in excess, removal of the 5′-dRP group from the substrate was dependent on the enzyme concentration (Figure 4A and B).Figure 3.


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)

Kinetic measurements of dRP lyase activity of Pol θ. The loss of dRP from the nicked AP-site containing DNA substrate was examined as a function of incubation time, and the rate of dRP removal by Pol θ was determined under single turnover conditions (i.e. enzyme/DNA = 4). The reaction was performed with Pol θ as described under Materials and Methods section. (A) Schematic representation of the DNA substrate prepared by annealing a 15-mer oligonucleotide, a 17-mer oligonucleotide containing 32P-labeled uracil at the 5′-end, and a 34-mer oligonucleotide template. (B) The duplex DNA (100 nM) was pretreated with UDG and then reacted with 400 nM Pol θ. Aliquots were withdrawn at the indicated time intervals and stabilized by the addition of 20 mM NaBH4 and incubation on ice for 30 min. Then, an equal volume of gel-loading dye buffer was added, and the reaction products were separated by precast 15% TBE-Urea gels. A representative phosphorimage of the polyacrylamide gel illustrates product analysis at various times (lanes 1–5) with and without Pol θ. (C) Time course data were analyzed using ImageQuant software and fitted to an exponential equation by using nonlinear least squares methods to determine the reaction rate (kobs= 0.14/min).
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Related In: Results  -  Collection

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Figure 3: Kinetic measurements of dRP lyase activity of Pol θ. The loss of dRP from the nicked AP-site containing DNA substrate was examined as a function of incubation time, and the rate of dRP removal by Pol θ was determined under single turnover conditions (i.e. enzyme/DNA = 4). The reaction was performed with Pol θ as described under Materials and Methods section. (A) Schematic representation of the DNA substrate prepared by annealing a 15-mer oligonucleotide, a 17-mer oligonucleotide containing 32P-labeled uracil at the 5′-end, and a 34-mer oligonucleotide template. (B) The duplex DNA (100 nM) was pretreated with UDG and then reacted with 400 nM Pol θ. Aliquots were withdrawn at the indicated time intervals and stabilized by the addition of 20 mM NaBH4 and incubation on ice for 30 min. Then, an equal volume of gel-loading dye buffer was added, and the reaction products were separated by precast 15% TBE-Urea gels. A representative phosphorimage of the polyacrylamide gel illustrates product analysis at various times (lanes 1–5) with and without Pol θ. (C) Time course data were analyzed using ImageQuant software and fitted to an exponential equation by using nonlinear least squares methods to determine the reaction rate (kobs= 0.14/min).
Mentions: To determine kinetic parameters of 5′-dRP group removal by Pol θ, we prepared a 34-bp duplex DNA that contained uracil at position 16 and a nick between positions 15 and 16, by annealing both a 15-mer and a 5′-end 32P-labeled uracil-containing 17-mer to the 34-mer complementary DNA strand. The resulting 32P-labeled nicked DNA was pretreated with UDG to generate the 5′-dRP-containing single-nucleotide gapped substrate; the DNA substrate, thus prepared, contained a 32P-labeled dRP flap in a single-nucleotide gap (Figure 3A, 16-mer + 32P-dRP). The rate of dRP removal by Pol θ was determined under single turnover conditions (i.e. enzyme/DNA = 4). The results revealed that Pol θ cleaved 5′ dRP in a time-dependent manner as monitored by disappearance of the substrate (Figure 3B), with an observed rate of dRP removal of ∼0.14/min (Figure 3C). In other experiments where substrate was in excess, removal of the 5′-dRP group from the substrate was dependent on the enzyme concentration (Figure 4A and B).Figure 3.

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