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AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe.

Nilsen L, Forstrøm RJ, Bjørås M, Alseth I - Nucleic Acids Res. (2011)

Bottom Line: AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases.A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate.These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Oslo University Hospital HF Rikshospitalet, PO Box 4950 Nydalen, N-0424 Oslo, Norway.

ABSTRACT
Abasic (AP) sites are formed spontaneously and are inevitably intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair initiated by DNA glycosylases performing β,δ-elimination cleavage of the AP sites has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites in Schizosaccharomyces pombe that is initiated by a bifunctional DNA glycosylase, Nth1 and followed by cleavage of the baseless sugar residue by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap. A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate. These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment.

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Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. (A) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1− (RHP357) and tdp1−nth1− (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. (B) Udg activity in the nth1− and tdp1−nth1− extracts. The nth1− and tdp1−nth1− extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
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gkr933-F2: Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. (A) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1− (RHP357) and tdp1−nth1− (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. (B) Udg activity in the nth1− and tdp1−nth1− extracts. The nth1− and tdp1−nth1− extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.

Mentions: To verify that Tdp1 was responsible for the observed 3′-P cleavage product of the nicked AP substrate, a tdp1−nth1− double mutant (RHP378) was generated. Total protein extract from RHP378 (tdp1−nth1−) and also RHP357 (nth1−) cells were prepared and used in activity assay with an Nth-nicked ds AP substrate. The result showed that the 3′-P cleavage product was absent in the double mutant (Figure 2A), confirming that Tdp1 possesses the major δ-elimination activity in S. pombe. To exclude that the missing 3′-P cleavage product was due to an inactive protein extract, the extracts from both nth1− and tdp1−nth1− cells were analyzed for an independent DNA repair activity and a uracil DNA glycosylase (Udg) assay was performed. Udg is a monofunctional DNA glycosylase removing uracils from DNA. The two extracts were near to identical for Udg activity, confirming that both extracts were functional (Figure 2B).Figure 2.


AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe.

Nilsen L, Forstrøm RJ, Bjørås M, Alseth I - Nucleic Acids Res. (2011)

Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. (A) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1− (RHP357) and tdp1−nth1− (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. (B) Udg activity in the nth1− and tdp1−nth1− extracts. The nth1− and tdp1−nth1− extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkr933-F2: Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. (A) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1− (RHP357) and tdp1−nth1− (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. (B) Udg activity in the nth1− and tdp1−nth1− extracts. The nth1− and tdp1−nth1− extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
Mentions: To verify that Tdp1 was responsible for the observed 3′-P cleavage product of the nicked AP substrate, a tdp1−nth1− double mutant (RHP378) was generated. Total protein extract from RHP378 (tdp1−nth1−) and also RHP357 (nth1−) cells were prepared and used in activity assay with an Nth-nicked ds AP substrate. The result showed that the 3′-P cleavage product was absent in the double mutant (Figure 2A), confirming that Tdp1 possesses the major δ-elimination activity in S. pombe. To exclude that the missing 3′-P cleavage product was due to an inactive protein extract, the extracts from both nth1− and tdp1−nth1− cells were analyzed for an independent DNA repair activity and a uracil DNA glycosylase (Udg) assay was performed. Udg is a monofunctional DNA glycosylase removing uracils from DNA. The two extracts were near to identical for Udg activity, confirming that both extracts were functional (Figure 2B).Figure 2.

Bottom Line: AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases.A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate.These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Oslo University Hospital HF Rikshospitalet, PO Box 4950 Nydalen, N-0424 Oslo, Norway.

ABSTRACT
Abasic (AP) sites are formed spontaneously and are inevitably intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair initiated by DNA glycosylases performing β,δ-elimination cleavage of the AP sites has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites in Schizosaccharomyces pombe that is initiated by a bifunctional DNA glycosylase, Nth1 and followed by cleavage of the baseless sugar residue by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap. A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate. These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment.

Show MeSH
Related in: MedlinePlus