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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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BER of AP sites in S. pombe. AP sites in S. pombe are mainly repaired by the short-patch BER pathway (thick arrows), initiated by incision of the AP site by Nth1, leaving 5′-P and 3′-dRP ends. The lethal 3′-block is further processed to a 3′-OH by the 3′-phosphodiester activity of Apn2. A DNA polymerase and a DNA ligase finally complete the repair by filling the gap and sealing the nick. Alternatively, Apn2 initiates repair by incision of the AP site leaving a 5′-dRP end, which can be further processed by long-patch (strand displacement synthesis and subsequent cleavage of the 5′-flap by Rad2) or short-patch BER (gap insertion and 5′-dRP lyase by SpPol4). Apn2 participates in both long-patch and short-patch BER. In this study, we propose an AP endonuclease independent branch of BER in S. pombe with Tdp1 working as a backup of Apn2 in the repair of 3′-dRP termini left after Nth1 cleavage. Pnk1 is suggested to work downstream of Tdp1 by processing the 3′-P generated by Tdp1, leaving a 3′-OH. Finally, repair is completed by a DNA polymerase and a DNA ligase.
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gkr933-F5: BER of AP sites in S. pombe. AP sites in S. pombe are mainly repaired by the short-patch BER pathway (thick arrows), initiated by incision of the AP site by Nth1, leaving 5′-P and 3′-dRP ends. The lethal 3′-block is further processed to a 3′-OH by the 3′-phosphodiester activity of Apn2. A DNA polymerase and a DNA ligase finally complete the repair by filling the gap and sealing the nick. Alternatively, Apn2 initiates repair by incision of the AP site leaving a 5′-dRP end, which can be further processed by long-patch (strand displacement synthesis and subsequent cleavage of the 5′-flap by Rad2) or short-patch BER (gap insertion and 5′-dRP lyase by SpPol4). Apn2 participates in both long-patch and short-patch BER. In this study, we propose an AP endonuclease independent branch of BER in S. pombe with Tdp1 working as a backup of Apn2 in the repair of 3′-dRP termini left after Nth1 cleavage. Pnk1 is suggested to work downstream of Tdp1 by processing the 3′-P generated by Tdp1, leaving a 3′-OH. Finally, repair is completed by a DNA polymerase and a DNA ligase.

Mentions: In summary, our results show that Tdp1 plays a more general role in DNA repair than only removal of Top1-mediated DNA damage. Tdp1 can be placed in a new branch of the BER pathway (Figure 5) working downstream of Nth1 by processing the 3′-α,β-unsaturated aldehyde left after Nth1 cleavage. A 3′-P terminus is generated by the Tdp1 reaction and S. pombe Pnk1 has been reported to have both 5′-kinase and 3′-phosphatase activity (40). Pnk1 is thus a likely candidate for processing of the 3′-P terminus to generate a 3′-OH. Repair can now be completed by a DNA polymerase filling in a new base and a DNA ligase sealing the nick. The tdp1− mutant was not hypersensitive to MMS as observed for the apn2− mutant, indicating that the Apn2-dependent pathway is the preferred one, with Tdp1 working as an important backup of Apn2 in the repair of AP sites.Figure 5.


AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe.

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

BER of AP sites in S. pombe. AP sites in S. pombe are mainly repaired by the short-patch BER pathway (thick arrows), initiated by incision of the AP site by Nth1, leaving 5′-P and 3′-dRP ends. The lethal 3′-block is further processed to a 3′-OH by the 3′-phosphodiester activity of Apn2. A DNA polymerase and a DNA ligase finally complete the repair by filling the gap and sealing the nick. Alternatively, Apn2 initiates repair by incision of the AP site leaving a 5′-dRP end, which can be further processed by long-patch (strand displacement synthesis and subsequent cleavage of the 5′-flap by Rad2) or short-patch BER (gap insertion and 5′-dRP lyase by SpPol4). Apn2 participates in both long-patch and short-patch BER. In this study, we propose an AP endonuclease independent branch of BER in S. pombe with Tdp1 working as a backup of Apn2 in the repair of 3′-dRP termini left after Nth1 cleavage. Pnk1 is suggested to work downstream of Tdp1 by processing the 3′-P generated by Tdp1, leaving a 3′-OH. Finally, repair is completed by a DNA polymerase and a DNA ligase.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr933-F5: BER of AP sites in S. pombe. AP sites in S. pombe are mainly repaired by the short-patch BER pathway (thick arrows), initiated by incision of the AP site by Nth1, leaving 5′-P and 3′-dRP ends. The lethal 3′-block is further processed to a 3′-OH by the 3′-phosphodiester activity of Apn2. A DNA polymerase and a DNA ligase finally complete the repair by filling the gap and sealing the nick. Alternatively, Apn2 initiates repair by incision of the AP site leaving a 5′-dRP end, which can be further processed by long-patch (strand displacement synthesis and subsequent cleavage of the 5′-flap by Rad2) or short-patch BER (gap insertion and 5′-dRP lyase by SpPol4). Apn2 participates in both long-patch and short-patch BER. In this study, we propose an AP endonuclease independent branch of BER in S. pombe with Tdp1 working as a backup of Apn2 in the repair of 3′-dRP termini left after Nth1 cleavage. Pnk1 is suggested to work downstream of Tdp1 by processing the 3′-P generated by Tdp1, leaving a 3′-OH. Finally, repair is completed by a DNA polymerase and a DNA ligase.
Mentions: In summary, our results show that Tdp1 plays a more general role in DNA repair than only removal of Top1-mediated DNA damage. Tdp1 can be placed in a new branch of the BER pathway (Figure 5) working downstream of Nth1 by processing the 3′-α,β-unsaturated aldehyde left after Nth1 cleavage. A 3′-P terminus is generated by the Tdp1 reaction and S. pombe Pnk1 has been reported to have both 5′-kinase and 3′-phosphatase activity (40). Pnk1 is thus a likely candidate for processing of the 3′-P terminus to generate a 3′-OH. Repair can now be completed by a DNA polymerase filling in a new base and a DNA ligase sealing the nick. The tdp1− mutant was not hypersensitive to MMS as observed for the apn2− mutant, indicating that the Apn2-dependent pathway is the preferred one, with Tdp1 working as an important backup of Apn2 in the repair of AP sites.Figure 5.

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