We examined BER capacity in the context of living cells using an assay system where Chroma-Luciferase expression representing synthesis of a reporter gene product is continuously measured. This approach has the benefit of providing a large number of data points on luciferase production with the economy of a single culture well. Use of this assay made it possible for us to observe subtle time-dependent changes in expression of luciferase associated with the repair of a specific single lesion-containing plasmid DNA in mouse fibroblast cells. We used two types of base lesion with the aim of examining both SN-BER and LP-BER sub-pathways. We viewed the uracil-DNA plasmid as a substrate for predominantly SN-BER and the THF-DNA plasmid as a substrate for LP-BER. As expected, the repair capacity for both uracil-DNA and THF-DNA was found to be lower in Pol β null cells compared with that in wild type cells (); yet, in all of our experiments, the measured reduction in repair in Pol β null cells was never more than ~50%. This reflected a high level of residual repair activity in the absence of Pol β, and the finding was surprising to us, based on earlier reports indicating that Pol β is the major DNA polymerase in SN-BER responsible for BER gap-filling synthesis and dRP lyase activity [9
The residual repair in the absence of Pol β could be as a result of back-up DNA polymerases, back-up DNA repair pathways, or an intrinsic background in our assay system. First, regarding the latter possibility, there are reports on the effects of abasic site lesions on RNA polymerase II (RNAPII) transcription involving experiments with purified systems and cell extracts [42
]. The abasic site lesion has been found to be a block for RNAPII [43
], and the single strand break generated from the conversion of these damaged sites by DNA glycosylase and/or APE1 activities blocks RNAPII transcription elongation [44
]. In our system, however, even if transcription occurs by lesion bypass before repair, the effects on the residual activity will be minimal because of the use of a stop codon and the rare C and U misinsertions opposite any bypass of the THF lesion (see ). Hence, this possible explanation for the residual repair in the absence of Pol β seems unlikely.
Next, concerning the possibility of back-up DNA polymerases, there appears to be a number of options available to the cell. DNA polymerase λ (Pol λ) is an X family polymerase, like Pol β, and is known to have the DNA polymerase and dRP lyase activities required for SN-BER [46
]. Although kinetic analyses demonstrated that the DNA synthesis turnover number of Pol λ is lower than that of Pol β, it seems clear that Pol λ can play a back-up role for Pol β, and furthermore, expression of Pol λ is strong in the mouse fibroblasts used here [49
]. Pol λ interacts with PCNA [51
], suggesting it may be involved in LP-BER as well as SN-BER, even when uracil-DNA is the substrate. In addition, DNA polymerases ι and θ have been suggested to function in BER [52
], and DNA polymerase δ and/or ε have been suggested to participate specifically in LP-BER [55
]. Accordingly, several DNA polymerases could compensate for a repair deficiency in the absence of Pol β, especially since the amount of damage used in the plasmid-based luciferase assay is expected to be lower than that following treatment of cells with most commonly used genotoxic agents. Nevertheless, despite the presence of these potential back-up polymerases, repair in Pol β null cells was not as strong as in wild type cells.
Finally, regarding the possibility that repair pathways other than BER participate in the Pol β-independent residual repair activity observed, mismatch repair does not seem to be a strong candidate for uracil/T, one reason that this mispair was chosen. The T/T mismatch is known to be a relatively poor substrate for mismatch repair [56
]. Based on the structural similarity between uracil and T, we expect this also to be a poor mismatch repair substrate. Transcription-coupled repair (TCR), initially described as a sub-pathway of nucleotide excision repair, also has been suggested to act on non-bulky base lesions like oxidative base damage [42
]. Hence, TCR may function to back-up repair in the Pol β-deficient mouse cells used here [63
]. Nevertheless, a better understanding of the Pol β-independent BER activity observed in these experiments will require further study.
In the presence of a PARP inhibitor, the repair capacity of wild type cells was decreased, and this reduction was to the same level as that found in Pol β null cells without inhibitor (). Moreover, the PARP inhibitor did not affect repair capacity in Pol β null cells. This indicates that PARP and Pol β activities are involved in the same repair process. Although not unexpected, these results are the first observation that the repair capacity for defined BER substrates is decreased by PARP inhibition in vivo. In contrast, cell sensitivity experiments have indicated that PARP activity plays a strong protective role in the response of replicating cells to alkylating agent-induced genotoxic stress, but this effect on cell sensitivity is not epistatic with the protective role of Pol β (i.e., the effects were additive). Taken together, it appears that Pol β and PARP may be partners in BER, as shown here, but that PARP activity has additional, Pol β-independent, roles in cellular protection against alkylating agent-induced damage.
Our data also demonstrated no differences in luciferase expression between the uracil-DNA plasmid and the positive control plasmid in wild type cells. This indicated rapid repair of uracil-DNA in wild type cells (). In contrast, the repair of THF-DNA in wild type cells was slower (). In the case of THF-DNA, the repair capacity in Pol β null cells expressing recombinant Pol β was higher than that of wild type cells, reflecting a situation where over-expression of Pol β can facilitate LP-BER to a level greater than that seen in wild type cells (). Thus, the level in wild type cells may be limiting for the type of LP-BER measured, since the Pol β level in wild type cells was lower than in Pol β-comp cells ().
It is noteworthy that we did not find differential effects of PARP inhibition on SN-BER versus LP-BER in these experiments. Favaudon et al. [67
] reported that the LP-BER sub-pathway was not affected by the absence of PARP-1 expression, whereas the SN-BER sub-pathway was affected by the deficiency. This interpretation was based on in vivo
recruitment analysis at sites of single strand breaks and did not include direct measurements of DNA repair. The role of PARP-1 and PARP activity in BER is still not fully understood, however, the results of our experiments are consistent with a scheme where PARP-1 and/or other PARP isoforms first bind to protect the nicked AP-site BER intermediate, then becomes automodified and leaves DNA allowing subsequent Pol β-dependent DNA synthesis. The effects of inhibition of PARP activity are in sharp contrast to a deficiency in PARP-1 expression [24
There are many versions of “unique lesion” plasmid-based assay systems for measurement of DNA repair (or lesion by-pass) in vivo. However, most of these assay systems involve preparation of large amounts of plasmid containing unique damage and the use of extracts from multiple culture wells to measure a reporter gene product. In these systems, the reporter signals from well to well are normalized by use of another plasmid, but cell conditions may not be identical from one culture well to the next. A notable difference between the system described here and previous systems is that continuous measurement allows use of a minimal amount of plasmid, and signal tracking within the same culture well allows more precision in measurements. For future advancement of this system, it may be possible to measure reported gene products from both a positive control plasmid and the lesion-containing plasmid within the same culture well by using signals that are measured at different wavelengths; this should provide even more accurate repair comparisons. Finally, in previous plasmid assays, repair efficiency generally was calculated with much longer culture periods after transfection (e.g., 24 or 48 h), whereas the present system involved use of a shorter culture period and comparison of repair in the log phase of cell growth.
In summary, we identified a reduction in cellular base lesion repair as a result of Pol β deficiency by making use of lesion-containing plasmids, along with a precise reporter gene expression assay. There was high residual repair activity in the absence of Pol β, probably due to back-up DNA polymerases and/or repair systems. Inhibition of cellular PARP activity resulted in a similar reduction in the repair capacity of wild type cells. The results highlight the existence of redundant BER pathways that require a better understanding before the BER capacity of a cell can be predicted or modulated. This assay system should facilitate future comparison of repair capacity in wild type and repair protein-deficient cells, and also testing of designed small molecule repair inhibitors that may alter repair capacity. In addition, it should be possible to evaluate repair of various other lesions mimicking those introduced by endogenous stress.