Single strand breaks SSBs encompass the disintegration of a single nucleotide and damaged 5 and or 3 termini. 9 Typical sources of SSBs include spontaneous hydrolysis leading to the formation of basic or apurinic apyrimidinic AP sites and further hydrolysis and oxidative damage by endogenous reactive oxygen species. ROS and ionizing radiation 10 SSBs can also be generated as intermediates of Endonuclease VIII like NEIL mediated BER and they can also arise as a result of chemical inhibition of cellular enzymes such as Top I 2 11 15. IR and free radicals generate two primary end groups at the 3 termini phosphate and phosphoglycolate whereas the primary end group at the 5 ends is phosphate 16. On the other hand damage by Top I inhibitors generate 3 phosphate and 5 OH termini. The removal of damaged bases by DNA glycosylases that possess an AP lyase activity generates 3 phosphate and 5 phosphate termini. 16 SSBs are considered a serious threat to genetic stability and cell survival If not repaired rapidly. Moreover, SSBs occur at a substantially higher frequency than double-strand breaks DSBs 17.
Most SSBs are repaired by a global SSBR process that can be divided into four basic steps SSB detection, DNA end processing, DNA gap filling and DNA ligation. i SSB detection is primarily mediated by poly ADP ribose polymerase 1 PARP 1 and PARP 2 which are critical for the recruitment of X-ray repair cross-complementing group. 1 protein XRCC1 and PNKP 17 20 XRCC1 is a scaffold protein required for the assembly of the other proteins at the site of DNA damage within the cell such as PNKP DNA polymerase β Pol β and DNA ligase III Lig 3. It has been shown previously that XRCC1 can stimulate both the 3 phosphatase and 5 kinase activities of PNKP and this stimulation is triggered by phosphorylation of a cluster of three CK2 phosphorylation sites at Ser518 Thr519 Thr523 in XRCC1 and high-affinity binding to the forkhead associated FHA domain of PNKP 21 24.
Non phosphorylated XRCC1 can also stimulate PNKP activity through a lower affinity interaction with the catalytic domain of PNKP and recent data have led to speculation that in cells XRCC1 is initially tethered to PNKP through the phosphorylation-dependent interaction followed by a shift to the low-affinity interaction to stimulate activity 25 26 ii DNA end processing of the 3 and or 5 termini which are often modified must be carried out to restore the correct/ 3 OH and 5 phosphate required for gap filling and DNA ligation to occur. As mentioned earlier PNKP plays critical roles in processing the damaged ends iii. The DNA gap is filled once damaged termini have been restored to their suitable configuration. Gap filling can occur by two different mechanisms. The insertion of the single nucleotide that is missing at most SSBs short patch repair can be catalyzed by Pol β. But at some SSBs gap filling may continue for multiple nucleotides and this is done by Pol δ Ɛ long patch repair 27. iv DNA ligation is the final step of SSBR Lig1 and Lig3 α appears to be the enzymes of choice during nuclear long patch repair and short patch SSBR respectively 28 30 but there is some controversy regarding the role of Lig3 in the repair of nuclear DNA. Lig3 is present in both the nucleus and mitochondria. A study has shown that mitochondrial Lig3 is required for cellular viability independent of the XRCC1 repair pathway 31. Figure 1 2 shows a model depicting the steps involved in the BER pathways.
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