We have utilized a model lesion consisting of two closely opposed furans to determine whether clustered lesions can be converted to DSBs in mammalian cells and to examine the involvement of NHEJ in preventing DSB formation. The damage was placed within the firefly luciferase coding region of a plasmid and nucleofected into cells. We determined that luciferase activity was significantly decreased by all types of clustered furans tested. This indicates that a DSB can be formed from two closely opposed lesions in a plasmid even in wild-type cells where NHEJ proteins are available for repair.
Initiation of repair of a furan is performed by a class II AP endonuclease, which introduces a strand break on the 5′ side of the damage. There are two class II AP endonucleases in mammalian cells: Ape1 and Ape2. Even though Ape1 has greater AP endonuclease activity than Ape2 in vitro
), both enzymes have been implicated in DSB formation from opposing AP sites during immunoglobulin class switch recombination in B cells (32
). Due to the higher AP endonuclease activity of Ape1, the mouse Ape1 (Apex1) was the likely candidate for causing the formation of the DSBs at the clustered furans in the mouse fibroblasts. Using siRNA we were able to implicate Apex1 in the formation of the DSB, but since we were still able to detect breakage when Apex1 was reduced to 20% its normal level, it is possible that the mouse Ape2 can also cleave at the clustered lesion, especially under conditions of reduced Apex1.
The level of luciferase activity measured from the clustered lesion constructs varied with the distance between the lesions: 12 bps apart > 2 bps apart > 5 bps apart. These results therefore indicate that less cleavage or greater repair occurred when the furans were 2 bps apart compared to 5 bps apart. In vitro
studies examining only the initiation of repair have shown that a SSB can inhibit the removal of a lesion if it is situated in the opposite strand ≤3bps away (6
), and that removal of one AP site can inhibit the removal of an opposing AP site, especially when it is situated 2 bps away and 5′ from the target AP site (10
). The lower formation of DSBs at the furans separated by 2 bps could therefore be due to Apex1 only cleaving at one furan in the cluster, generating a SSB-repair intermediate that prevents further action of Apex1 at the second furan. When the distance between the furans increased to 5 bps apart, the inhibition was lost/decreased and initiation of repair occurred at both furans, resulting in greater formation of DSBs and a further decrease in luciferase activity. Previous in vitro
experiments have shown greater cleavage of lesions separated by ≥5 bps (6
Increasing the lesions to 12 bps apart decreased the formation of DSBs as seen by the increase in firefly luciferase activity. This is likely due to an increase in the complete repair of the two single strand breaks introduced at the furans. It is possible that the hydrogen bonding created by the intervening 12 bps stabilized the DNA structure, decreasing the chance of DSB formation prior to complete repair. This is in agreement with our work on clustered lesions in bacteria, where using a similar assay and two opposing uracils we determined that moving the lesions from 7 to 13 bps apart increased luciferase activity and plasmid recovery (26
). A similar situation in bacteria was also seen for two opposing furans situated 5 and 20 bps apart (27
In wild-type cells all the proteins were available to repair DSBs and so the luciferase activity measured from the constructs could have been partially due to accurate repair of a fraction of the DSBs generated from the initiation of clustered lesion repair. Gulston et al.
) implicated NHEJ in repair of ~10% of the non-DSB clusters produced by γ-rays in Chinese hamster ovary cells, and suggested that these lesions may consist of two closely opposed AP sites. In vitro
studies have also shown that the Ku70/80 protein can inhibit base excision repair by binding to an opposing SSB (34
). We therefore examined repair in both Ku80−/−
mouse fibroblasts. The activity of the furan constructs in these NHEJ-defective cells was very similar to that in the isogenic wild-type cells. This indicates that Ku80 did not disrupt the cleavage of the furans in the cluster, and that NHEJ is not involved in the accurate repair of the clustered lesions. It is unlikely that this negative result was due to a lack of activation of the DNA repair pathways, even though these experiments were performed without pre-treatment of the cells with ionizing radiation. Ionizing radiation is known to generate DSBs and result in a cascade of signaling events that include DNA-PK and ATM activation (35
). However, even the addition of linear DNA to cell extracts has been found to activate ATM (37
) and damage-containing plasmids electroporated into cells can stimulate non-homologous as well as homologous recombination (38
). Since our experiments use ligations that contain linear DNA as well as damaged circular DNA, it is likely that the presence of DSBs activates the repair pathways. Future experiments are planned to determine whether the outcome of the clustered lesions is altered by irradiation of the cells prior to nucleofection of the damaged plasmid.
NHEJ is known to repair DSBs inaccurately (20
). Re-isolation of the plasmid DNA from WT1 cells showed that there was ~45% recovery of plasmid DNA containing furans separated by 2 or 5 bps, even though these lesions resulted in much lower luciferase activities compared to the undamaged sequence. This suggested that a portion of the plasmid did not contain the accurate luciferase coding sequence and was generated by inaccurate repair. These same constructs resulted in ~4 times less recovery of plasmid compared to the undamaged sequence from cells that were deficient in Ku80. This indicated that NHEJ or Ku80 in the wild-type cells was required for the inaccurate repair of the clustered lesions and prevention of DNA degradation.
PCR analysis did confirm that a fraction of the repair products contained deletions and fewer deletions were present in DNA isolated from Ku80−/−
cells. Sequencing of the products revealed that deletions/inaccurate repair had frequently occurred at regions containing microhomology sequences, where one copy of the microhomology was deleted upon formation of the junction. Since deletions were detected in the Ku80−/−
cells, these must have been generated by Ku80-independent inaccurate repair of the strand breaks at the clustered lesions. Previous repair studies using Ku80-deficient cell lines have identified DNA containing deletions with junctions at microhomology regions (39
). This type of deleted product can be generated by microhomology-mediated end-joining (MMEJ) as well as single strand annealing (SSA; 40). Our data therefore implicates MMEJ and SSA in the repair of DSBs generated at clustered lesions. Ku80 is also believed to be involved in MMEJ: Ku80-deficient Chinese hamster ovary cells showed ~75% reduction in MMEJ (22
). This may explain why this type of product was detected in this study in wild-type cells and appeared to be reduced in the small number of samples processed from Ku80−/−
cells. SSA requires Rad52, while Fen1 has been found to be involved in MMEJ (40
). Parp-1 and DNA ligase III have also been implicated in a ‘back-up’ end-joining mechanism in the absence of functional DNA-PK mediated NHEJ (41
). Although it is beyond the scope of this study, it will be interesting to examine the contributions of Rad52, Fen1, Parp-1 and DNA ligase III to the repair of clustered furans in both wild-type and Ku80−/−
cells by the use of siRNA.
In summary (), we have shown that clustered lesions consisting of two closely opposed furans can be converted to a DSB in wild-type cells and we have implicated Apex1 in mouse cells as the enzyme responsible for the initiation of repair and DSB formation. The formation of the DSB, escape from DSB repair and plasmid degradation was dependent on the distance separating the two lesions. Our studies indicate that NHEJ does not play a role in accurate repair of clustered lesions, but this work implicates Ku80 and hence NHEJ in the inaccurate repair of DSBs generated by Apex1 at clustered abasic sites. We also detected Ku80-independent repair that used microhomology sequences to join the termini, so implicating MMEJ and SSA in the repair of clustered lesions.
Figure 4. Repair of clustered lesions. Two opposing furans (F) can be removed from the plasmid DNA by Apex1 and potentially Ape2, causing the generation of two single strand breaks (SSBs) that can interact to generate a double strand break (DSB). The 5′ (more ...)
This work, using a model system where the clustered damage is situated in a plasmid, demonstrates that clustered lesions are biologically relevant damage, and suggests that the cell will attempt to repair clustered lesions in chromosomal DNA both accurately and inaccurately. However, we have also shown that repair is not always complete resulting in mutagenic lesions being converted to potentially lethal DSBs. This has implications for future development of adjuvant therapies for radiotherapy. Our work suggests that not only would it be beneficial to enhance AP endonuclease repair in irradiated tumors, but that inhibition of NHEJ would prevent the repair of AP endonuclease-generated DSBs and enhance cell killing.
Future work will focus on identifying the proteins involved in repair in cells and the biological consequences of more complex clustered lesions, which in vitro
) have been shown to severely compromise repair.