Here, we confirm the importance of both XRCC1 and PARP in the repair induced by oxidative damage to the DNA (A, D and E). XRCC1 is necessary for repair after induction of oxidative damage to the DNA, most likely by recruiting and stabilizing Ligase IIIα (19
). PARP inhibition also has a decelerating effect on the rate of ligation, but the two proteins clearly have distinct roles in this repair, as PARP inhibition further delay SSB repair in XRCC1 defective cells. We find that more than half of the H2
-induced SSBs are repaired within 5
min after damage induction in the reconstituted wild-type cells (D), showing that this repair is a fast process. It should be noted that this repair includes the BER of oxidized bases as well as SSB repair, as H2
produces a variety of lesions to the DNA (21
). However, we see a characteristic rapid repair curve where the majority of SSBs are present in the cells directly after treatment and not after enzymatically-induced incisions, as can be seen after treatment with DMS. Thus, we suggest that the majority of the breaks originate from direct SSBs.
We also find that XRCC1 defective cells are hypersensitive to PARP inhibitors (B) in spite of the fact that there was no statistically significant increase of the background levels of SSBs in these cells (C), suggesting that the increased sensitivity of XRCC1 defective cells to PARP inhibitors is explained by functions of PARP1 and XRCC1 in processes apart from SSB repair. For instance, blocked replication forks activates PARP1 for efficient restart to occur (22
) and both PARP1 and XRCC1 have been shown to have specific roles at replication forks in S phase cells (8
). In speculation, there is a possibility that inhibition of PARP would generate a toxic lesion at replication forks that requires XRCC1 for repair, or vice versa. Also, it should be noted that we have not observed any indications of this hypersensitivity affecting the cells viability during the short time of treatment and repair that are employed in this study.
After confirming the importance of functional XRCC1 and PARP in the repair of SSBs, we move on to compare and evaluate the role of PARP in the repair of alkylated DNA damages. By utilizing XRCC1 deficient cells and a PARP inhibitor, we efficiently block the ligation of DNA breaks and thus we can monitor early repair events. After DMS treatment we find a high level of SSBs in XRCC1 deficient cells as well as in PARP inhibited cells, suggesting that neither XRCC1 nor PARP are necessary for the detection or excision of alkylated DNA damages repaired by BER (B and C). Additionally, we see even higher levels of SSBs when impeding both proteins simultaneously by inhibiting PARP in XRCC1 deficient cells, leading to the conclusion that the inhibition of PARP does not affect the repair of DMS induced damages in the same manner as XRCC1 deficiency does.
We confirm that the subsequent SSBs induced by DMS predominantly originate from base alkylations, which are detected by N-methylpurine-DNA glycosylase (MPG). The level of SSBs is considerably reduced when MPG is knocked down by siRNA depletion (B). Importantly, as no or very few SSBs are formed after DMS treatment in MPG siRNA depleted cells, our data demonstrate that MPG is the main (and possibly the only) glycosylase to recognize and excise DMS-induced base damage from DNA.
The role of PARP1 is likely to recognize SSBs or other DNA ends in the DNA and to increase the repair. We find that H2
-induced SSB repair is retarded in cells when treated with a PARP inhibitor, as a likely consequence of reduced recruitment of XRCC1 (8
). Furthermore, we find that siRNA depletion of PARP1 did not affect the amount of DMS-induced SSBs, or if anything decreased the amount of SSBs (B). This can be explained by PARP1 not having a direct role in BER. In the literature, PARP1 is denoted as a BER protein, which is likely related to previous observations that cell extracts from PARP1−/−
cells are unable to complete the DNA synthesis step of BER in vitro
). However, the presence of PARP1 is not required for effective BER in an in vitro
biochemical assay, and may even decrease the biochemical kinetics of BER (24
). So far, it has not been possible to assign an exact function of PARP1 in BER (24
), but our data support a model where BER does not require PARP1. This is also in line with PARP1 protein being poorly conserved while many other BER factors are well conserved through evolution. The model proposes that the subset of BER SSB intermediates that become uncoupled somewhere during the repair pathway, are bound by PARP1 when it is present in the cell (). When PARP is inhibited it is thereby trapped on these SSB intermediates, thus blocking their ligation and causing a potentially toxic retention of SSBs in the DNA. This would explain the increase of DMS-induced SSBs observed following PARP inhibition. Such trapping of PARP to SSB intermediates may explain the high toxicity of PARP inhibitors in BRCA2 defective cells, in contrast to the very modest toxicity of co-depletion of BRCA2 and PARP1 (25
Figure 7. Model for BER and the influence of PARP. The MPG glycosylase is recognizing N-methylated purines, likely through scanning the DNA for base lesions. Once a lesion is recognized, MPG excises the damaged base and the APE1 endonuclease cleaves the newly formed (more ...)
There are two competing models for BER. One model separate the BER incision and the subsequent SSB repair, meaning that the DNA lesion would be identified twice; first by a glycosylase and then by PARP1 for SSB repair. The other model suggests that the DNA lesion is only recognized by a glycosylase and all following steps are coordinated () (26–28
). Since we find no accumulation of the SSB intermediate in PARP1 siRNA depleted cells, we suggest that the DNA lesion is only identified by glycosylases/endonucleases and that the following steps are coordinated.
BER of DMS induced damages appears to be a fast and efficient repair pathway as the majority of the SSB intermediates are incised and ligated within 30
min after exposure to the DNA damaging agent (B, B and A). However, we find that a large number of repair events (as measured by SSB formation after addition of a PARP inhibitor) occur in the cells 2 h after DMS was removed from the medium (), which suggests that there is still ongoing repair at this time. According to our model, the repair events we detect through PARP inhibition represents only a fraction of uncoupled SSB intermediates, indicating that an even higher amount of total BER events still occur in the cell at this time. We speculate that this ongoing repair could be the result of slow damage detection by the glycosylase, possibly because of inaccessibility to the location of the lesion or the complexity of the lesion. We also see a difference in cells with siRNA depleted MPG compared to wild-type cells, indicating that we are indeed studying actual BER events (D). However, it should be noted that there is no certain way to ensure that the damages repaired after 2
h are in fact original damages that were produced at the time of treatment. There is a possibility that cellular DMS is not removed in the washing step and that this could sit in the cell and make damage 2.25
h after initiation of the treatment. Even in this scenario DMS hydrolyzes in aqueous solutions with a half life of 17
min at 37°C (29
), so only 0.3% of the original DMS would remain in the cells, even if no DMS was removed by the washing step. Our conclusion is that it takes time for the BER machinery to identify a fraction of the damaged bases to make the incisions.