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DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions.

Parsons JL, Preston BD, O'Connor TR, Dianov GL - Nucleic Acids Res. (2007)

Bottom Line: We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions.Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta).Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire, UK.

ABSTRACT
Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIalpha and DNA polymerase beta. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta). Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol delta are partially deficient in the repair of these 'complex' lesions, demonstrating the importance of Pol delta during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation.

Show MeSH
Repair ability of mouse embryonic fibroblasts deficient in Pol δ exonuclease against Hx located as a second nucleotide 5′- to a DNA single strand break. Plasmid constructs used in the experiments were prepared by ligation of the shown oligonucleotide duplexes into a luciferase reporter vector (A). Plasmids were transfected into either wild-type (Pol δ +/+) or mutant (Pol δ D400A) cells and after 8 h cell extracts were prepared and luciferase activity was measured. The average and standard error are shown graphically (B). Asterix (*) represents a statistical difference P < 0.02 compared to the wild-type cell line using t test.
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Figure 8: Repair ability of mouse embryonic fibroblasts deficient in Pol δ exonuclease against Hx located as a second nucleotide 5′- to a DNA single strand break. Plasmid constructs used in the experiments were prepared by ligation of the shown oligonucleotide duplexes into a luciferase reporter vector (A). Plasmids were transfected into either wild-type (Pol δ +/+) or mutant (Pol δ D400A) cells and after 8 h cell extracts were prepared and luciferase activity was measured. The average and standard error are shown graphically (B). Asterix (*) represents a statistical difference P < 0.02 compared to the wild-type cell line using t test.

Mentions: The pGL3-control luciferase reporter vector (Promega) was digested with Hind III and Nco I restriction enzymes to remove a sequence of 33 bp between the promoter and luciferase gene. The plasmid was electrophoresed on a 0.7% agarose gel and the linear plasmid DNA isolated from the gel using the QIAquick gel extraction kit (Qiagen). Oligonucleotides were 5′-phosphorylated using T4 polynucleotide kinase and duplexes were constructed (Figure 8A) by heating to 90°C for 3–5 min followed by slow cooling to room temperature. A 200 μl ligation reaction containing 1 μg (300 fmol) linear plasmid DNA, 1.5 pmol oligonucleotide duplex and 40 U T4 DNA ligase in buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 25 μg/ml BSA was incubated overnight at 4°C. Analysis by agarose gel electrophoresis demonstrated that approximately 10% of the plasmid DNA was circularized with oligonucleotide duplex during the ligation reaction. The DNA was concentrated using Amicon-30 columns (Millipore) and buffer exchanged by washing twice with 1 mM Tris-HCl (pH 8.0), prior to concentration to 10 μl. Pol δ D400A exonuclease deficient and the corresponding wild-type cell lines were seeded at 1 × 105 cells/well in a volume of 500 μl in a 24-well plate and cultured overnight at 37°C in 5% CO2. The crude DNA ligation reactions, in combination with 0.2 μg pRL-TK renilla luciferase reporter vector, used as an internal control, were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer using a 1:1 ratio of plasmid (μg) to reagent (μl) in a volume of 100 μl. The cells were incubated further for 8 h at 37°C in 5% CO2 and cell extracts prepared by washing the cells in PBS prior to the addition of 100 μl passive lysis buffer (Promega) and incubation at room temperature for 15 min with agitation. The extracts were collected and frozen at −70°C until required. For measurement of firefly and renilla luciferase expression, the dual-luciferase reporter assay system (Promega) and a luminoskan ascent luminometer (Thermo) were used as described by the manufacturers. The results were expressed as a ratio of firefly to renilla luciferase expression and an average ratio was obtained in each experiment from duplicate transfections. These values were then expressed as a percentage against the average ratio for the undamaged sequence and then normalized to that obtained with the wild-type cell line that was set to 100%. At least five transfection experiments were performed for each substrate prepared.


DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions.

Parsons JL, Preston BD, O'Connor TR, Dianov GL - Nucleic Acids Res. (2007)

Repair ability of mouse embryonic fibroblasts deficient in Pol δ exonuclease against Hx located as a second nucleotide 5′- to a DNA single strand break. Plasmid constructs used in the experiments were prepared by ligation of the shown oligonucleotide duplexes into a luciferase reporter vector (A). Plasmids were transfected into either wild-type (Pol δ +/+) or mutant (Pol δ D400A) cells and after 8 h cell extracts were prepared and luciferase activity was measured. The average and standard error are shown graphically (B). Asterix (*) represents a statistical difference P < 0.02 compared to the wild-type cell line using t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851633&req=5

Figure 8: Repair ability of mouse embryonic fibroblasts deficient in Pol δ exonuclease against Hx located as a second nucleotide 5′- to a DNA single strand break. Plasmid constructs used in the experiments were prepared by ligation of the shown oligonucleotide duplexes into a luciferase reporter vector (A). Plasmids were transfected into either wild-type (Pol δ +/+) or mutant (Pol δ D400A) cells and after 8 h cell extracts were prepared and luciferase activity was measured. The average and standard error are shown graphically (B). Asterix (*) represents a statistical difference P < 0.02 compared to the wild-type cell line using t test.
Mentions: The pGL3-control luciferase reporter vector (Promega) was digested with Hind III and Nco I restriction enzymes to remove a sequence of 33 bp between the promoter and luciferase gene. The plasmid was electrophoresed on a 0.7% agarose gel and the linear plasmid DNA isolated from the gel using the QIAquick gel extraction kit (Qiagen). Oligonucleotides were 5′-phosphorylated using T4 polynucleotide kinase and duplexes were constructed (Figure 8A) by heating to 90°C for 3–5 min followed by slow cooling to room temperature. A 200 μl ligation reaction containing 1 μg (300 fmol) linear plasmid DNA, 1.5 pmol oligonucleotide duplex and 40 U T4 DNA ligase in buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 25 μg/ml BSA was incubated overnight at 4°C. Analysis by agarose gel electrophoresis demonstrated that approximately 10% of the plasmid DNA was circularized with oligonucleotide duplex during the ligation reaction. The DNA was concentrated using Amicon-30 columns (Millipore) and buffer exchanged by washing twice with 1 mM Tris-HCl (pH 8.0), prior to concentration to 10 μl. Pol δ D400A exonuclease deficient and the corresponding wild-type cell lines were seeded at 1 × 105 cells/well in a volume of 500 μl in a 24-well plate and cultured overnight at 37°C in 5% CO2. The crude DNA ligation reactions, in combination with 0.2 μg pRL-TK renilla luciferase reporter vector, used as an internal control, were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer using a 1:1 ratio of plasmid (μg) to reagent (μl) in a volume of 100 μl. The cells were incubated further for 8 h at 37°C in 5% CO2 and cell extracts prepared by washing the cells in PBS prior to the addition of 100 μl passive lysis buffer (Promega) and incubation at room temperature for 15 min with agitation. The extracts were collected and frozen at −70°C until required. For measurement of firefly and renilla luciferase expression, the dual-luciferase reporter assay system (Promega) and a luminoskan ascent luminometer (Thermo) were used as described by the manufacturers. The results were expressed as a ratio of firefly to renilla luciferase expression and an average ratio was obtained in each experiment from duplicate transfections. These values were then expressed as a percentage against the average ratio for the undamaged sequence and then normalized to that obtained with the wild-type cell line that was set to 100%. At least five transfection experiments were performed for each substrate prepared.

Bottom Line: We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions.Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta).Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire, UK.

ABSTRACT
Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIalpha and DNA polymerase beta. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta). Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol delta are partially deficient in the repair of these 'complex' lesions, demonstrating the importance of Pol delta during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation.

Show MeSH