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Poly(ADP-ribose) polymerase-2 (PARP-2) activity contributes to a cells' poly(ADP-ribosyl)ating potential and like PARP-1, has been implicated in several DNA repair pathways including base excision repair and DNA single strand break repair. Here the consequences of its stable depletion in HeLa, U20S, and AS3WT2 cells were examined. All three PARP-2 depleted models showed increased sensitivity to the cell killing effects on ionizing radiation as reported in PARP-2 depleted mouse embryonic fibroblasts providing further evidence for a role in DNA strand break repair. The PARP-2 depleted HeLa cells also showed both higher constitutive and DNA damage-induced levels of polymers of ADP-ribose (PAR) associated with unchanged PARP-1 protein levels, but higher PARP activity and a concomitant lower PARG protein levels and activity. These changes were accompanied by a reduced maximal recruitment of PARP-1, XRCC1, PCNA, and PARG to DNA damage sites. This PAR-associated phenotype could be reversed in HeLa cells on re-expression of PARP-2 and was not seen in U20S and AS3WT2 cells. These results highlight the complexity of the relationship between different members of the PARP family on PAR metabolism and suggest that cell model dependent phenotypes associated with the absence of PARP-2 exist within a common background of radiation sensitivity.

DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5(KD) cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5(KD) cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5(KD) compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5(KD) cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5(KD) cells is due to a role of Cdk5 in other pathways or the altered polymer levels.