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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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In vitro BER with purified proteins. Gap-filling activity of Pol θ was evaluated on a uracil-containing BER substrate by measuring incorporation of [α-32P]dCMP as a function of incubation time and protein concentration. Reaction conditions and product analysis were as described under Materials and Methods section. A 35-bp oligonucleotide duplex DNA (250 nM) with a U:G mismatch was mixed with 15 nM UDG, 15 nM APE, 200 nM DNA ligase I, 400 or 800 nM purified 98-kDa Pol θ and incubated at 37°C. Aliquots were withdrawn at indicated time intervals, and an equal volume of gel-loading buffer was added to terminate the reaction. The reaction products were separated by 15% denatured PAGE, and a PhosphorImager was used to scan the gel. (A) Phosphorimage of denaturing PAGE demonstrating the in vitro BER activity of Pol θ is shown. The migration position of the ligated BER product is indicated. (B) BER products were quantified using ImageQuant software, and the relative phophorImager units were plotted against time of incubation.
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Figure 7: In vitro BER with purified proteins. Gap-filling activity of Pol θ was evaluated on a uracil-containing BER substrate by measuring incorporation of [α-32P]dCMP as a function of incubation time and protein concentration. Reaction conditions and product analysis were as described under Materials and Methods section. A 35-bp oligonucleotide duplex DNA (250 nM) with a U:G mismatch was mixed with 15 nM UDG, 15 nM APE, 200 nM DNA ligase I, 400 or 800 nM purified 98-kDa Pol θ and incubated at 37°C. Aliquots were withdrawn at indicated time intervals, and an equal volume of gel-loading buffer was added to terminate the reaction. The reaction products were separated by 15% denatured PAGE, and a PhosphorImager was used to scan the gel. (A) Phosphorimage of denaturing PAGE demonstrating the in vitro BER activity of Pol θ is shown. The migration position of the ligated BER product is indicated. (B) BER products were quantified using ImageQuant software, and the relative phophorImager units were plotted against time of incubation.

Mentions: Since the 98-kDa Pol θ was able to cleave the 5′-dRP flap from a BER intermediate and also retained DNA polymerase activity, we asked whether the 98-kDa enzyme was capable of supporting BER in vitro. We assembled repair reaction mixtures containing a 35-bp DNA substrate with uracil opposite guanine and purified human enzymes, including UDG, AP endonuclease, DNA ligase I and the 98-kDa Pol θ. Reactions were initiated by the addition of Mg2+ and [α-32P]dCTP, and repair activity was monitored by the incorporation of [α-32P]dCMP in place of dUMP into a ligated 35-bp DNA product as a function of incubation time and concentration of Pol θ (Figure 7). In vitro repair activity was dependent on both enzyme concentration and incubation time (Figure 7A and B), confirming that human Pol θ catalyzed sufficient dRP removal and 1-nt gap filling to support single-nucleotide BER.Figure 7.


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)

In vitro BER with purified proteins. Gap-filling activity of Pol θ was evaluated on a uracil-containing BER substrate by measuring incorporation of [α-32P]dCMP as a function of incubation time and protein concentration. Reaction conditions and product analysis were as described under Materials and Methods section. A 35-bp oligonucleotide duplex DNA (250 nM) with a U:G mismatch was mixed with 15 nM UDG, 15 nM APE, 200 nM DNA ligase I, 400 or 800 nM purified 98-kDa Pol θ and incubated at 37°C. Aliquots were withdrawn at indicated time intervals, and an equal volume of gel-loading buffer was added to terminate the reaction. The reaction products were separated by 15% denatured PAGE, and a PhosphorImager was used to scan the gel. (A) Phosphorimage of denaturing PAGE demonstrating the in vitro BER activity of Pol θ is shown. The migration position of the ligated BER product is indicated. (B) BER products were quantified using ImageQuant software, and the relative phophorImager units were plotted against time of incubation.
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Figure 7: In vitro BER with purified proteins. Gap-filling activity of Pol θ was evaluated on a uracil-containing BER substrate by measuring incorporation of [α-32P]dCMP as a function of incubation time and protein concentration. Reaction conditions and product analysis were as described under Materials and Methods section. A 35-bp oligonucleotide duplex DNA (250 nM) with a U:G mismatch was mixed with 15 nM UDG, 15 nM APE, 200 nM DNA ligase I, 400 or 800 nM purified 98-kDa Pol θ and incubated at 37°C. Aliquots were withdrawn at indicated time intervals, and an equal volume of gel-loading buffer was added to terminate the reaction. The reaction products were separated by 15% denatured PAGE, and a PhosphorImager was used to scan the gel. (A) Phosphorimage of denaturing PAGE demonstrating the in vitro BER activity of Pol θ is shown. The migration position of the ligated BER product is indicated. (B) BER products were quantified using ImageQuant software, and the relative phophorImager units were plotted against time of incubation.
Mentions: Since the 98-kDa Pol θ was able to cleave the 5′-dRP flap from a BER intermediate and also retained DNA polymerase activity, we asked whether the 98-kDa enzyme was capable of supporting BER in vitro. We assembled repair reaction mixtures containing a 35-bp DNA substrate with uracil opposite guanine and purified human enzymes, including UDG, AP endonuclease, DNA ligase I and the 98-kDa Pol θ. Reactions were initiated by the addition of Mg2+ and [α-32P]dCTP, and repair activity was monitored by the incorporation of [α-32P]dCMP in place of dUMP into a ligated 35-bp DNA product as a function of incubation time and concentration of Pol θ (Figure 7). In vitro repair activity was dependent on both enzyme concentration and incubation time (Figure 7A and B), confirming that human Pol θ catalyzed sufficient dRP removal and 1-nt gap filling to support single-nucleotide BER.Figure 7.

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