PARP1 suppresses homologous recombination events in mice in vivo

Abstract

Recent studies suggest that PARP1 inhibitors, several of which are currently in clinical trial, may selectively kill BRCA1/2 mutant cancers cells. It is thought that the success of this therapy is based on immitigable lethal DNA damage in the cancer cells resultant from the concurrent loss or inhibition of two DNA damage repair pathways: single-strand break (SSB) repair and homologous recombination repair (HRR). Presumably, inhibition of PARP1 activity obstructs the repair of SSBs and during DNA replication, these lesions cause replication fork collapse and are transformed into substrates for HRR. In fact, several previous studies have indicated a hyper-recombinogenic phenotype in the absence of active PARP1 in vitro or in response to DNA damaging agents. In this study, we demonstrate an increased frequency of spontaneous HRR in vivo in the absence of PARP1 using the pun assay. Furthermore, we found that the HRR events that occur in Parp1 nullizygous mice are associated with a significant increase in large, clonal events, as opposed to the usually more frequent single cell events, suggesting an effect in replicating cells. In conclusion, our data demonstrates that PARP1 inhibits spontaneous HRR events, and supports the model of DNA replication transformation of SSBs into HRR substrates.

A Conditional Mouse Model for Measuring the Frequency of Homologous Recombination Events In Vivo in the Absence of Essential Genes

“…Further, it should be noted that PARP1 inhibition is synthetic lethal with BRCA1 deficiency (29), most likely due to the lack of PARP1-dependent single-strand break repair, leading to increased stalled replication forks that cannot be repaired in a BRCA1 HR repair-dependent manner. Considering these previous observations and the results from this study in combination with the study by Claybon et al, we hypothesize that p un revertant multicell events are the outcome of HR events tied to replication and that at least a majority of the single-cell events are not (26). Furthermore, these replication-associated HR events are likely to be RAD51 dependent, whereas replication-independent HR resulting in p un reversion is likely the result of a RAD51/BRCA1-independent SSA event.…”

“…This synthetic lethal approach is premised on the hypothesis that the inability to repair damaged DNA by HR (e.g., in many BRCA1/2 mutant cancers) forces the cell to rely upon alternative mechanisms and, in the absence of such alternatives (e.g., resulting from PARP1 inhibition), will lead to cellular death. Recently, as shown in a paper by Claybon et al, our laboratory used the p un HR model to show that the loss of PARP1 leads to a hyperrecombinagenic phenotype (26). This result, in combination with the present study, gives in vivo proof of concept for how PARP1 inhibition is an effective therapeutic against tumors displaying BRCAness (4).…”

“…Earlier studies using the p un assay report an average of ϳ5 to 6 eye spots per wild-type RPE (8,10). In more recent studies, our laboratory reported a similar frequency of eye spot events in wild-type samples (17,26 Fig. S1 in the supplemental material).…”

“…6B). For comparison, Claybon et al found that the increase of HR events in the absence of PARP1 was due primarily to large multicell events (26). Brca1 expression is induced prior to DNA synthesis and stays abundant during S and G 2 phase (21,41,70).…”

“…This development has been described as "radially outward from the optic nerve with an edge-biased pattern that has a churning motion" (10). Therefore, we can retrospectively define when these reversion events occur, similar to using the concentric circles for aging trees (7,17,26). As such, p un reversion events that occur close to the optic nerve must have arisen earlier in development than more distal events, which must have occurred later.…”