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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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Genetic interactions between tdp1− and BER mutants. (A) CPT sensitivity in the fission yeast tdp1− strain. Exponentially growing wild-type (FY526) and tdp1− (RHP379) cells were serial diluted in water and spotted onto YES plates containing CPT as indicated. The plates were incubated for 3 days at 30°C and the survival assessed. (B) MMS survival analysis of the tdp1− and BER single and double mutants. Exponentially growing wild-type (FY526), nth1− (RHP357), apn2− (RHP302), tdp1− (RHP379), nth1−apn2− (RHP383), tdp1−nth1− (RHP378), tdp1−apn2− (RHP382) and tdp1−apn2−nth1− (RHP381) cells were serial diluted in water and spotted onto YES plates containing MMS as indicated. The plates were incubated for 3 days at 30°C and the survival assessed.
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gkr933-F4: Genetic interactions between tdp1− and BER mutants. (A) CPT sensitivity in the fission yeast tdp1− strain. Exponentially growing wild-type (FY526) and tdp1− (RHP379) cells were serial diluted in water and spotted onto YES plates containing CPT as indicated. The plates were incubated for 3 days at 30°C and the survival assessed. (B) MMS survival analysis of the tdp1− and BER single and double mutants. Exponentially growing wild-type (FY526), nth1− (RHP357), apn2− (RHP302), tdp1− (RHP379), nth1−apn2− (RHP383), tdp1−nth1− (RHP378), tdp1−apn2− (RHP382) and tdp1−apn2−nth1− (RHP381) cells were serial diluted in water and spotted onto YES plates containing MMS as indicated. The plates were incubated for 3 days at 30°C and the survival assessed.

Mentions: Processing of the 3′-dRP terminus generated by Nth1 cleavage of an AP site, suggests a role of Tdp1 in the BER pathway acting downstream of Nth1. To investigate this, a tdp1− single mutant (RHP379) was constructed. In addition, double and triple mutants of tdp1− and BER mutant cells were generated; RHP378 (tdp1−nth1−), RHP381 (tdp1−apn2−nth1−) and RHP382 (tdp1−apn2−). The anticancer drug camptothecin (CPT) acts by stabilizing Top1–DNA adducts, resulting in strand breaks in the DNA (37). Tdp1 is shown to be the key enzyme for removal of such adducts (24,38). When challenged with CPT, fission yeast cells lacking Tdp1 became sensitive as previously reported (Figure 4A) (39). Next, the tdp1− mutant was exposed to the alkylating agent MMS that via the Mag1 DNA glycosylase generates a heavy burden of AP sites. The tdp1− single mutant displayed no sensitivity to MMS and the tdp1−nth1− double mutant was as sensitive as the nth1− single mutant (Figure 4B). This is in accordance with Nth1 and Tdp1 acting in the same pathway. Further, lack of sensitization of the nth1− mutant by deletion of tdp1, confirmed our biochemical data that showed lack of a robust AP lyase activity of S. pombe Tdp1. In contrast, the tdp1−apn2− double mutant showed a synergistic increase in MMS sensitivity compared to the respective single mutants (Figure 4B), suggesting that Tdp1 and Apn2 act on the same substrate. In absence of both Tdp1 and Apn2, the 3′-blocking lesion (3′-dRP) produced by Nth1 is hardly processed and contributes to the cytotoxicity seen in the tdp1−apn2−mutant after MMS exposure. The importance of these two genes was also evident under normal conditions as the tdp1−apn2− double mutant was more slowly growing (Figure 4B, left panel). Deletion of nth1 in a tdp1−apn2− background partially relieved the MMS sensitivity of the tdp1−apn2− double mutant (Figure 4B). This is similar to what happens to the apn2− single mutant when nth1 is deleted [Figure 4B, and (28)]. This is probably due to accumulation of 3′-blocking lesions generated by Nth1 that cannot be further processed in the absence of Apn2 and Tdp1. 3′-dRP termini are more toxic to the cell than intact AP sites, which can be bypassed by translesions polymerases. The observed slow growing phenotype of the tdp1−apn2− double mutant in unstressed cells appears to be relieved in the tdp1−apn2−nth1− triple mutant (Figure 4B, left panel), demonstrating that Nth1 also acts on endogenously generated DNA damage.Figure 4.


AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe.

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

Genetic interactions between tdp1− and BER mutants. (A) CPT sensitivity in the fission yeast tdp1− strain. Exponentially growing wild-type (FY526) and tdp1− (RHP379) cells were serial diluted in water and spotted onto YES plates containing CPT as indicated. The plates were incubated for 3 days at 30°C and the survival assessed. (B) MMS survival analysis of the tdp1− and BER single and double mutants. Exponentially growing wild-type (FY526), nth1− (RHP357), apn2− (RHP302), tdp1− (RHP379), nth1−apn2− (RHP383), tdp1−nth1− (RHP378), tdp1−apn2− (RHP382) and tdp1−apn2−nth1− (RHP381) cells were serial diluted in water and spotted onto YES plates containing MMS as indicated. The plates were incubated for 3 days at 30°C and the survival assessed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3300018&req=5

gkr933-F4: Genetic interactions between tdp1− and BER mutants. (A) CPT sensitivity in the fission yeast tdp1− strain. Exponentially growing wild-type (FY526) and tdp1− (RHP379) cells were serial diluted in water and spotted onto YES plates containing CPT as indicated. The plates were incubated for 3 days at 30°C and the survival assessed. (B) MMS survival analysis of the tdp1− and BER single and double mutants. Exponentially growing wild-type (FY526), nth1− (RHP357), apn2− (RHP302), tdp1− (RHP379), nth1−apn2− (RHP383), tdp1−nth1− (RHP378), tdp1−apn2− (RHP382) and tdp1−apn2−nth1− (RHP381) cells were serial diluted in water and spotted onto YES plates containing MMS as indicated. The plates were incubated for 3 days at 30°C and the survival assessed.
Mentions: Processing of the 3′-dRP terminus generated by Nth1 cleavage of an AP site, suggests a role of Tdp1 in the BER pathway acting downstream of Nth1. To investigate this, a tdp1− single mutant (RHP379) was constructed. In addition, double and triple mutants of tdp1− and BER mutant cells were generated; RHP378 (tdp1−nth1−), RHP381 (tdp1−apn2−nth1−) and RHP382 (tdp1−apn2−). The anticancer drug camptothecin (CPT) acts by stabilizing Top1–DNA adducts, resulting in strand breaks in the DNA (37). Tdp1 is shown to be the key enzyme for removal of such adducts (24,38). When challenged with CPT, fission yeast cells lacking Tdp1 became sensitive as previously reported (Figure 4A) (39). Next, the tdp1− mutant was exposed to the alkylating agent MMS that via the Mag1 DNA glycosylase generates a heavy burden of AP sites. The tdp1− single mutant displayed no sensitivity to MMS and the tdp1−nth1− double mutant was as sensitive as the nth1− single mutant (Figure 4B). This is in accordance with Nth1 and Tdp1 acting in the same pathway. Further, lack of sensitization of the nth1− mutant by deletion of tdp1, confirmed our biochemical data that showed lack of a robust AP lyase activity of S. pombe Tdp1. In contrast, the tdp1−apn2− double mutant showed a synergistic increase in MMS sensitivity compared to the respective single mutants (Figure 4B), suggesting that Tdp1 and Apn2 act on the same substrate. In absence of both Tdp1 and Apn2, the 3′-blocking lesion (3′-dRP) produced by Nth1 is hardly processed and contributes to the cytotoxicity seen in the tdp1−apn2−mutant after MMS exposure. The importance of these two genes was also evident under normal conditions as the tdp1−apn2− double mutant was more slowly growing (Figure 4B, left panel). Deletion of nth1 in a tdp1−apn2− background partially relieved the MMS sensitivity of the tdp1−apn2− double mutant (Figure 4B). This is similar to what happens to the apn2− single mutant when nth1 is deleted [Figure 4B, and (28)]. This is probably due to accumulation of 3′-blocking lesions generated by Nth1 that cannot be further processed in the absence of Apn2 and Tdp1. 3′-dRP termini are more toxic to the cell than intact AP sites, which can be bypassed by translesions polymerases. The observed slow growing phenotype of the tdp1−apn2− double mutant in unstressed cells appears to be relieved in the tdp1−apn2−nth1− triple mutant (Figure 4B, left panel), demonstrating that Nth1 also acts on endogenously generated DNA damage.Figure 4.

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