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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.

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Purification of the major activity against 5-OHU located as a second nucleotide 5′- to a DNA single strand break in human cells. A protein purification scheme was designed using HeLa cells (A) and involved generating whole cell extracts (step 1), separation of proteins by phosphocellulose chromatography using a step elution of 0.15 M KCl (PC-FI) and 1 M KCl (PC-FII; step 2), separation of proteins in PC-FII by Superose 12 gel-filtration chromatography (step 3), followed by separation of proteins (>100 kDa) using a Mono-Q column and a gradient elution of 0.05–1 M KCl (step 4). During each stage, the activity against a 5′-labelled 5-OHU lesion located as a second nucleotide 5′- to a DNA single strand break (5-OHU2) was measured and the corresponding active fractions pooled, dialysed if necessary and used in the following step. Activity against 5-OHU2 from fractions eluted from the final Mono-Q stage are shown (B) and these were further analysed by western blotting using XPF and Pol δ specific antibodies.
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Figure 4: Purification of the major activity against 5-OHU located as a second nucleotide 5′- to a DNA single strand break in human cells. A protein purification scheme was designed using HeLa cells (A) and involved generating whole cell extracts (step 1), separation of proteins by phosphocellulose chromatography using a step elution of 0.15 M KCl (PC-FI) and 1 M KCl (PC-FII; step 2), separation of proteins in PC-FII by Superose 12 gel-filtration chromatography (step 3), followed by separation of proteins (>100 kDa) using a Mono-Q column and a gradient elution of 0.05–1 M KCl (step 4). During each stage, the activity against a 5′-labelled 5-OHU lesion located as a second nucleotide 5′- to a DNA single strand break (5-OHU2) was measured and the corresponding active fractions pooled, dialysed if necessary and used in the following step. Activity against 5-OHU2 from fractions eluted from the final Mono-Q stage are shown (B) and these were further analysed by western blotting using XPF and Pol δ specific antibodies.

Mentions: To isolate the major excision activity against DNA lesions located as a second nucleotide 5′-upstream to a SSB, a protein purification scheme was designed using the 5-OHU2 substrate to monitor excision activity (Figure 4A). Briefly, HeLa WCE was generated by the method of Manley et al. (24) and proteins subsequently separated by phosphocellulose chromatography using a step elution of buffers containing 0.15 M (PC-FI) and 1 M KCl (PC-FII). An excision activity against 5-OHU2 was detected in PC-FII that was subsequently fractionated by gel filtration chromatography on a Superose 12 column. The major peak of activity corresponded to a protein of molecular weight greater than 100 kDa using protein standards (data not shown). The peak activity fractions were pooled, dialysed and separated on a Mono-Q column using an elution gradient of 50–1000 mM KCl. Fractions containing excision activity against the 5-OHU2 substrate were subsequently detected, eluting at approximately 250 mM KCl (Figure 4B). What was apparent from the activity profile was that the enzyme may be an exonuclease due to the cleavage of at least five sequential nucleotides. The major known exonucleases in human cells include DNA polymerase δ (Pol δ), DNA polymerase ɛ, Werner (WRN) protein and MRE11 (29). However, recent evidence has suggested that xeroderma pigmentosum group F complementing protein (XPF) may be involved in the removal of 3′-blocked termini from DNA strand breaks (30). We therefore probed active fractions for XPF protein and found that it co-purified, although not completely aligned, with the activity against the 5-OHU2 substrate (Figure 4C). However, immunodepletion of active fractions using XPF antibodies or analysing the activity of purified ERCC1–XPF complex on the 5-OHU2 substrate, known to be active on a stem-loop containing substrate, did not ablate the activity (data not shown). As the 3′-5′ exonuclease activity of WRN protein has been shown to be inhibited by the presence of certain oxidative modifications (31) we eliminated this protein as the major activity against this substrate. We switched our attention to Pol δ and using western blotting and antibodies specific to this protein, similar to XPF protein, we observed Pol δ in active fractions (Figure 4C). Immunodepletion of Pol δ from the peak active fraction and subsequent analysis by western blotting revealed that the levels of Pol δ are only slightly reduced by mock immunodepletion compared to the original fraction, while the Pol δ specific antibodies completely removed the Pol δ protein from the fraction (Figure 5A). However, we also show that XPF remains in the mock- and Pol δ− immunodepleted fraction indicating the antibody specificity (Figure 5A). We further demonstrated that the mock-immunodepleted fraction has a slightly reduced excision activity against 5-OHU2 (Figure 5B), in accordance with the slightly reduced levels of Pol δ compared to the original fraction caused by the mock-immunodepletion protocol. Furthermore, immunodepletion using Pol δ specific antibodies ablates the activity observed in the purified Mono-Q fraction indicating that Pol δ is the major activity in human cell extracts eliminating 5-OHU located as a second nucleotide 5′-upstream to a SSB (Figure 5B).Figure 4.


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)

Purification of the major activity against 5-OHU located as a second nucleotide 5′- to a DNA single strand break in human cells. A protein purification scheme was designed using HeLa cells (A) and involved generating whole cell extracts (step 1), separation of proteins by phosphocellulose chromatography using a step elution of 0.15 M KCl (PC-FI) and 1 M KCl (PC-FII; step 2), separation of proteins in PC-FII by Superose 12 gel-filtration chromatography (step 3), followed by separation of proteins (>100 kDa) using a Mono-Q column and a gradient elution of 0.05–1 M KCl (step 4). During each stage, the activity against a 5′-labelled 5-OHU lesion located as a second nucleotide 5′- to a DNA single strand break (5-OHU2) was measured and the corresponding active fractions pooled, dialysed if necessary and used in the following step. Activity against 5-OHU2 from fractions eluted from the final Mono-Q stage are shown (B) and these were further analysed by western blotting using XPF and Pol δ specific antibodies.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 4: Purification of the major activity against 5-OHU located as a second nucleotide 5′- to a DNA single strand break in human cells. A protein purification scheme was designed using HeLa cells (A) and involved generating whole cell extracts (step 1), separation of proteins by phosphocellulose chromatography using a step elution of 0.15 M KCl (PC-FI) and 1 M KCl (PC-FII; step 2), separation of proteins in PC-FII by Superose 12 gel-filtration chromatography (step 3), followed by separation of proteins (>100 kDa) using a Mono-Q column and a gradient elution of 0.05–1 M KCl (step 4). During each stage, the activity against a 5′-labelled 5-OHU lesion located as a second nucleotide 5′- to a DNA single strand break (5-OHU2) was measured and the corresponding active fractions pooled, dialysed if necessary and used in the following step. Activity against 5-OHU2 from fractions eluted from the final Mono-Q stage are shown (B) and these were further analysed by western blotting using XPF and Pol δ specific antibodies.
Mentions: To isolate the major excision activity against DNA lesions located as a second nucleotide 5′-upstream to a SSB, a protein purification scheme was designed using the 5-OHU2 substrate to monitor excision activity (Figure 4A). Briefly, HeLa WCE was generated by the method of Manley et al. (24) and proteins subsequently separated by phosphocellulose chromatography using a step elution of buffers containing 0.15 M (PC-FI) and 1 M KCl (PC-FII). An excision activity against 5-OHU2 was detected in PC-FII that was subsequently fractionated by gel filtration chromatography on a Superose 12 column. The major peak of activity corresponded to a protein of molecular weight greater than 100 kDa using protein standards (data not shown). The peak activity fractions were pooled, dialysed and separated on a Mono-Q column using an elution gradient of 50–1000 mM KCl. Fractions containing excision activity against the 5-OHU2 substrate were subsequently detected, eluting at approximately 250 mM KCl (Figure 4B). What was apparent from the activity profile was that the enzyme may be an exonuclease due to the cleavage of at least five sequential nucleotides. The major known exonucleases in human cells include DNA polymerase δ (Pol δ), DNA polymerase ɛ, Werner (WRN) protein and MRE11 (29). However, recent evidence has suggested that xeroderma pigmentosum group F complementing protein (XPF) may be involved in the removal of 3′-blocked termini from DNA strand breaks (30). We therefore probed active fractions for XPF protein and found that it co-purified, although not completely aligned, with the activity against the 5-OHU2 substrate (Figure 4C). However, immunodepletion of active fractions using XPF antibodies or analysing the activity of purified ERCC1–XPF complex on the 5-OHU2 substrate, known to be active on a stem-loop containing substrate, did not ablate the activity (data not shown). As the 3′-5′ exonuclease activity of WRN protein has been shown to be inhibited by the presence of certain oxidative modifications (31) we eliminated this protein as the major activity against this substrate. We switched our attention to Pol δ and using western blotting and antibodies specific to this protein, similar to XPF protein, we observed Pol δ in active fractions (Figure 4C). Immunodepletion of Pol δ from the peak active fraction and subsequent analysis by western blotting revealed that the levels of Pol δ are only slightly reduced by mock immunodepletion compared to the original fraction, while the Pol δ specific antibodies completely removed the Pol δ protein from the fraction (Figure 5A). However, we also show that XPF remains in the mock- and Pol δ− immunodepleted fraction indicating the antibody specificity (Figure 5A). We further demonstrated that the mock-immunodepleted fraction has a slightly reduced excision activity against 5-OHU2 (Figure 5B), in accordance with the slightly reduced levels of Pol δ compared to the original fraction caused by the mock-immunodepletion protocol. Furthermore, immunodepletion using Pol δ specific antibodies ablates the activity observed in the purified Mono-Q fraction indicating that Pol δ is the major activity in human cell extracts eliminating 5-OHU located as a second nucleotide 5′-upstream to a SSB (Figure 5B).Figure 4.

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
Related in: MedlinePlus