Abstract
Upon DNA binding the poly(ADP-ribose) polymerase family of enzymes (PARPs) add multiple ADP-ribose subunits to themselves and other acceptor proteins. Inhibitors of PARPs have become an exciting and real prospect for monotherapy and as sensitizers to ionising radiation (IR). The action of PARPs are reversed by poly(ADP-ribose) glycohydrolase (PARG). Until recently studies of PARG have been limited by the lack of an inhibitor. Here, a first in class, specific, and cell permeable PARG inhibitor, PDD00017273, is shown to radiosensitize. Further, PDD00017273 is compared with the PARP1/2/3 inhibitor olaparib. Both olaparib and PDD00017273 altered the repair of IR-induced DNA damage, resulting in delayed resolution of RAD51 foci compared with control cells. However, only PARG inhibition induced a rapid increase in IR-induced activation of PRKDC (DNA-PK) and perturbed mitotic progression. This suggests that PARG has additional functions in the cell compared with inhibition of PARP1/2/3, likely via reversal of tankyrase activity and/or that inhibiting the removal of poly(ADP-ribose) (PAR) has a different consequence to inhibiting PAR addition. Overall, our data are consistent with previous genetic findings, reveal new insights into the function of PAR metabolism following IR and demonstrate for the first time the therapeutic potential of PARG inhibitors as radiosensitizing agents.
Highlights
The poly(ADP-ribose) polymerase (PARP) family of enzymes are recruited to, and activated at, sites of DNA damage, where they add poly(ADP-ribose) (PAR) to themselves and to other DNA repair and chromatin-remodeling factors [1,2]
Concomitant exposure to olaparib and PDD00017273 resulted in reduced PAR accumulation, confirming the specificity of the poly (ADP-ribose) glycohydrolase (PARG) inhibitor and the reagents
In the presence of the PARG inhibitor a large increase in PAR was observed using both techniques, suggesting that PAR synthesis is induced by ionising radiation (IR), but that the turnover is too rapid to always observe in control cells
Summary
The poly(ADP-ribose) polymerase (PARP) family of enzymes are recruited to, and activated at, sites of DNA damage, where they add poly(ADP-ribose) (PAR) to themselves and to other DNA repair and chromatin-remodeling factors [1,2]. Once synthesised the PAR polymer is thought to act as a signal to recruit repair factors to the damage In this way PARP proteins are considered to play a key role in coordinating the repair of single [3,4,5,6,7,8,9,10] and double strand DNA breaks [11,12,13,14,15], and in the restart of stalled or collapsed DNA replication forks [16,17,18]. Radiosensitization by these inhibitors is generally considered to be a replication dependent event [44,45]
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