PARP1 inhibitors regulate PARP1 structure independent of DNA, reducing binding affinity for single strand breaks
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Abstract
Cancers caused by mutations to the DNA repair machinery may be treated by inhibitors that target Poly(ADP-ribose) Polymerase 1 (PARP1). PARP inhibitors are thought to cause toxicity by trapping PARP1 at single strand breaks, preventing single strand break repair, thus leading to accumulation of DNA damage and cancer cell death. Intriguingly though, different PARP inhibitors display similar cellular toxicities and catalytic inhibition despite having widely varying trapping potencies. To better understand this apparent contradiction and identify complementary mechanisms of action, we here visualize the effect of inhibitors on individual PARP1 and PARP2 molecules by atomic force microscopy (AFM). We find, surprisingly, that inhibitors cause significant PARP1 compaction and loss of molecular flexibility also in the absence of DNA. This compaction correlates with the trapping potency of the inhibitor; and could be functionally relevant by reducing the subsequent binding of pre-treated PARP1 to DNA. Such changes are less pronounced for PARP2, which shares a high sequence identity with the PARP1 catalytic domain but lacks the DNA binding domain present in PARP1. Our findings reveal an additional, DNA-independent mechanism of action for PARP inhibitors, where PARP inhibitors with strong trapping potencies target PARP1 in the absence of DNA, compact their conformation and thereby reduce its ability to bind to DNA.
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Notably, forinhibitors causing the strongest compaction, olaparib and talazoparib, (see Fig. 1), exposingPARP1 to these inhibitors before DNA resulted in a 4 and 6-fold reduction, respectively, inthe subsequent binding to DNA, compared with protocols in which PARP1 was allowed tobind to DNA before exposure to inhibitors (Fig. 3B)
I was curious what is known about how PARP1 finds it's nicked DNA target? Is it possible that the reduced binding to the nicked substrate is due to a reduction in the efficiency of finding the substrate (i.e. sliding or inter- or intra-strand transfer?).
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Taken together with Figure 1, thisindicates that upon binding to talazoparib, PARP1 undergoes a large-scale compaction thatstabilises the PARP1 conformation.
This is a really elegant biochemical study--congratulations on the work! I was wondering if you could or have confirmed this compaction using an alternate methodology, like sedimentation velocity analytical ultracentrifugation?
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