The dynamics and regulation of PARP1 and PARP2 in response to DNA damage and during replication

Abstract

DNA strand breaks activate Poly(ADP-ribose) polymerase (PARP) 1 and 2, which use NAD+ as the substrate to covalently conjugate ADP-ribose on themselves and other proteins (e.g., Histone) to promote chromatin relaxation and recruit additional DNA repair factors. Enzymatic inhibitors of PARP1 and PARP2 (PARPi) are promising cancer therapy agents that selectively target BRCA1- or BRCA2- deficient cancers. As immediate early responders to DNA strand breaks with robust activities, PARP1 and PARP2 normally form transient foci (<10 minutes) at the micro-irradiation-induced DNA lesions. In addition to enzymatic inhibition, PARPi also extend the presence of PARP1 and PARP2 at DNA lesions, including at replication forks, where they may post a physical block for subsequent repair and DNA replication. The dynamic nature of PARP1 and PARP2 foci made live cell imaging a unique platform to detect subtle changes and the functional interaction among PARP1, PARP2, and their regulators. Recent imaging studies have provided new understandings of the biological consequence of PARP inhibition and uncovered functional interactions between PARP1 and PARP2 and new regulators (e.g., histone poly(ADP-ribosylation) factor). Here, we review recent advances in dissecting the temporal and spatial Regulation of PARP1 and PARP2 at DNA lesions and discuss their physiological implications on both cancer and normal cells.

Keywords: Live cell quantitative imaging; PAPR1; PARP2; PARylation; Replication.

Figure 1.. Diagram illustrating the behavior of PARP1 and PARP2 with or without treatment of PARP inhibitors.

(a) In the absence of PARPi, PARP1 and PARP2 act as sensors of DNA breaks. PARP1 and PAPR2 can disassociate from the breaks without auto-PARylation, which allows each break to activate multiple PARP1 and PARP2 molecules. Transient interaction with DNA ends activates PARP1 and PARP2, triggering auto-PARylation and PARylation of histones. Activation of PARP1 generates most of the PAR chains, which recruits XRCC-LIG3 and other DNA repair proteins to facilitate DNA repair. In the presence of PARPi, PARP1 continuously turns over at the unrepaired DNA damage sites, forming a dynamic yet apparently long-lasting focus. The lack of PAR delays the recruitment of XRCC1 and other repair proteins and causes the persistence of DNA breaks. In contrast, clinical PARPi delay the exchange of PARP2 significantly. Thus, each PARP1 stays longer at the breaks, causing accumulative increase of PARP2 intensity. (b) The diagram of FRAP recovery for PARP1 and PARP2. We note that PARP2 seems to exchange faster than PARP1. Clinical PARPi significantly delay PARP2 exchanges but moderately impact PARP1 exchanges. Since PARP1 is responsible for most DNA damage induced PARylation, the PARylation activity of PARP1 facilitates the recruitment of PARP2. Inhibiting PARP1 severely reduces DNA damage-induced PARylation and delays the repair, leading to continuous recruitment of different PARP1 to the damage sites. In this context, loss of PARP2 activity has a relatively minor impact on overall DNA damage induced PARylation.

Figure 2.. The multiple roles of PARPs at DNA replication fork.

(a) In otherwise normal cells, PARPs can act both upstream and downstream of replication forks. Downstream of the replication fork, unligated Okazaki fragments can activate PARPs, which generates PAR and recruits XRCC1-LIG3 as a backup pathway for Okazaki fragments maturation. Alternatively, template DNA strand breaks can activate PARPs to facilitate nick repair. In the absence of PARPs, the progression of the replication fork might convert a single-stranded break to a single-ended DSB, which will need either homology recombination or a converge fork to complete the replication. (b) PARPs maintain the regressed replication forks when the fork encounters DNA damage. PARP1 can either inhibit the recruitment of RecQ1 to restart the fork or recruit MRN complex and start the HR repair to restart the fork. While in the absence of PARP1 or under the treatment of PARPi, RecQ1 is recruited to restart the fork and induce DSB, which may subsequently lead to cell death. The absence of PARP1 would also increase the PrimPol induced translesion synthesis to bypass the DNA lesion.