PARP-1 is an abundant nuclear protein with diverse and wide-ranging functions in the cell. The ubiquitous presence of PARP-1 across various processes in DNA metabolism has complicated understanding the unique roles it plays in BER/SSB and DSB repair. Despite the ambiguity in its functions, it is clear that PARP-1 activation is important in the cellular response to DNA damage. PAR synthesis directly impacts protein recruitment to sites of DNA damage, it can regulate the efficiency of repair, and in some cases can even lead to cell death.
Here, we examined PAR levels associated with BER-proficiency (wild-type) and BER-deficiency (pol β null) in mouse fibroblast cells. The PAR level increased dramatically in pol β null cells after exposure to the DNA alkylating agent, MMS () and also after laser irradiation of cells in cell imaging experiments (). Using this micro-irradiation and imaging approach, we showed that the pol β inhibitor PA partially blocked pol β from being recruited to the sites of DNA damage; therefore, the increase in PAR associated with the presence of PA appeared to reflect a defect in pol β recruitment. From these data, we proposed that the elevated PAR level may act as a reporter of a deficiency in DNA repair. Consistent with this hypothesis, a study by Gottipati et. al., found that deficiencies in BRCA2 or shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3
all resulted in hyperactivation of PARP-1 
. Thus, PARylation may play a role in the amplification of DNA damage and repair signaling. Since the PARP-1 hyperactivation state in pol β null cells is transient and does not induce necrosis, it is probable that the role of the increased PARylation is to signal a deficiency in BER. In the absence of such a deficiency, there was no increase in PARylation after MMS treatment (). In addition to hyper-PARylation serving as an indicator for deficiency in BER, recruitment of DNA repair proteins by PAR has been well documented 
. We propose that hyperactivation of PARP-1 serves as the signal for recruitment of a back-up DNA repair system to the sites of damage, allowing for repair and increased cell viability in the absence of pol β.
PARP-1 is critical to DSB repair by the backup-NHEJ pathway. This backup pathway, involving PARP-1, XRCC1, and DNA ligase III, in concert with MRE11 and NBS1 (MRN) and possibly other as yet unidentified proteins, is responsible for the residual end-joining of DSBs in cells deficient in components of classical NHEJ and is independent of Ku70/Ku80 and DNA-PK 
. While the components and mechanisms of this pathway are still under study, it has been established that the kinetics of DSB repair are slower than classical-NHEJ 
and that it is repressed by Ku under normal conditions 
. Examination of the interaction of PARP-1 with Ku70 revealed increased association after MMS treatment, and an increased association of PAR-adducted Ku70 with PARP-1 in pol β null cells (). With this emerging picture about the relationship between PARP-1, PARylation, and Ku70, we aimed to verify a protective role of NHEJ in pol β null cells by utilizing the DNA-PK inhibitor, NU-7026, in combination with MMS treatment. Under these DNA-PK inhibited conditions, there was enhanced necrotic cell death of pol β null cells compared with wild-type cells. This increase suggested a link between PARP-1 hyperactivation and DNA-PK-dependent NHEJ in MMS-treated pol β-deficient cells.
During NHEJ, damaged DNA ends are captured by the Ku heterodimer (Ku70/Ku80) that recruits and activates DNA-PKcs, which in turn mediates the ligation of the DNA ends by the DNA ligase IV/XRCC4/XLF complex 
. Ku70 and DNA-PK together compose the key complex in classical-NHEJ. Ku70 competes for DNA binding with PARP-1 making classical-NHEJ the dominant pathway over PARP-1-dependent backup-NHEJ 
. In vitro
PARylation of Ku70 was shown to reduce its DNA-binding activity and to inhibit classical-NHEJ 
. In addition to covalent modification by PAR, Ku70 has a pADPr binding motif (a.a. 246–261) that may enhance its interaction with PARylated proteins 
. PARylation of the DNA-PK catalytic subunit by PARP-1 has been shown to enhance its kinase activity 
, supporting its stimulatory role in DSB repair 
. In addition, a recent study in Dictyostelium discoideum
demonstrated that the Ku70 PAR-binding zinc finger (PBZ) 
is required for recruitment to DSBs through PAR binding 
. While vertebrate Ku70 does not contain this PBZ domain, it does raise the question of the role PAR plays in the recruitment of vertebrate Ku70 to DSBs in vivo.
The pADPr motif of Ku70 may act to coordinate its interaction with PARylated DNA-PK as previously predicted 
, and with PARylated PARP-1 or other proteins at the DNA damage sites. Competition between Ku70 and PARP-1 has been shown most effectively in vitro
, but neither of these studies examined the PARylation state of PARP-1 or Ku70. Additionally, Ku represses backup-NHEJ under normal conditions 
making the interplay between PARP-1, PARylation, and Ku70 an important target for further investigation.
Our findings using pol β null cells and MMS treatment initially appeared consistent with another study, which also noted an increase in PAR levels compared with wild-type cells. However, those authors attributed the increase in PAR as a cell death signal leading to PARP-1-mediated necrosis 
. In contrast, the dramatic increase in PAR observed here in pol β null cells within 10 min after MMS exposure was down-regulated to the untreated cellular level within 30 min after exposure. In order to verify that necrotic cell death was not initiated by depletion of intracellular ATP under our treatment conditions, we measured intracellular ATP levels (Figure S1
) and the fraction of necrotic cells 24 h after MMS exposure (). These data indicated that the observed PARP-1 hyperactivation in pol β null cells did not induce necrosis and failed to deplete ATP levels.
Taken together, our results suggest a model where BER deficiency leads to increased production of PAR by PARP-1, and this serves to amplify a damage signal while activating and recruiting downstream repair elements to address the DNA damage (). Whether the PARylated PARP-1 is maintained at the site of DNA damage by the toxic unrepaired BER intermediate 
or dissociates from the DNA when a PARylation maximum is reached is unclear 
. The decrease in PAR levels observed at 30 min, which coincides with the increasing interaction of Ku70 and PARP-1, leads us to propose that PARP-1 is likely retained at the site of damage to sequester toxic BER intermediates until significant amounts of Ku70 can be recruited. Ku70 then competes for DNA binding with PARP-1 leading to dissociation of PARP-1 from DNA. It is possible that Ku70 can utilize its dRP lyase activity 
to remove blocks to subsequent repair. How unrepaired BER intermediates are converted to DSBs is still unknown at this point. Possibly the extended remodeling of chromatin by PARP-1 at the sites of unrepaired damage assists this conversion. Once DSBs occur, classical-NHEJ ensues.
Model of PARP-1 hyperactivation and recruitment of NHEJ proteins.
While we cannot exclude other DNA repair mechanisms in cells when BER is defective, our evidence points to classical-NHEJ rather than backup-NHEJ being enlisted to rescue stalled base lesion repair intermediates. This link between PARP-1 hyperactivation and hand-off to classical-NHEJ provides evidence of cross-talk between the roles of PARP-1 in BER/SSB and DSB repair.