Poly(ADP-Ribose) Polymerase 1 Accelerates Single-Strand Break Repair in Concert with Poly(ADP-Ribose) Glycohydrolase

Abstract

Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.Single-strand breaks (SSBs) are the commonest type of lesion arising in cells and can arise from direct attack of deoxyribose, as abortive intermediates of topoisomerase 1 activity, or as normal intermediates of base excision repair. One of the earliest responses to DNA strand breakage is the induction of poly(ADP-ribose) (PAR) synthesis (reviewed in references 17 and 35). Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant and stable component of chromatin and is the major source of PAR synthesis following DNA strand breakage (27,40). PARP-1 rapidly binds to and is activated by DNA singleand double-strand breaks, resulting in covalent modification of itself and to a lesser extent other target proteins with long chains of PAR (4,5,15,41,42). The binding and activity of PARP-1 at DNA breaks are very transient because the ribosylated enzyme dissociates from DNA through charge repulsion (24, 60). Subsequently, a second DNA damage-activated PARP was identified in human cells and was called PARP-2 (1, 30). PARP-2 has 18-fold lower activity than PARP-1 but can support up to 25% of normal levels of DNA damage-induced PAR synthesis in the absence of PARP-1 (1, 49). While PARP-1 is the primary source of global PAR synthesis following DNA strand breakage, it is possible that PARP-2 fulfils an overlapping or backup role. In support of this, mice lacking either PARP-1 or PARP-2 are viable, but mice lacking both enzymes are not (38). The presence of high levels of PAR in cells following DNA strand breakage is very transient because the polymer is rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG). Consequently, proteins that become ribosylated following DNA strand breakage are rapidly converted back to their unmodified form (16,32,56,60). PARG is composed of a 110-kDa nuclear form and at least two cytoplasmic isoforms of 99 kDa and 103 kDa, each of which most likely arises from the same primary transcript (34, 39).Despite their central roles in PAR metabolism, the relative importance of PARP-1, ...

Coordinated Regulation of TIP60 and Poly(ADP-Ribose) Polymerase 1 in Damaged-Chromatin Dynamics

“…Presently, it is unclear which process of NBS1 accumulation is defective in PARP-1-deficient cells. However, in PARP-1-defective cells, the phosphorylation of H2AX reportedly is not abolished and actually is rather increased (38,39). Furthermore, in this study we also found that shPARP-1-expressing cells, or cells treated with olaparib, are defective in H2AX exchange at DNA damage sites.…”

Nonhomologous end-joining promotes resistance to DNA damage in the absence of an ADP-ribosyltransferase that signals DNA single strand breaks

“…Unfortunately, the inability to disrupt murine parp1 and parp2 in combination prohibits testing this model in defined genetic backgrounds (Ménissier de Murcia et al, 2003). However, depletion of PARP2 alone or in combination with PARP1 does not dramatically impact on the ability of human cells to repair DNA strand breaks (Fisher et al, 2007), suggesting ARTs other than PARP2 are responsible for signalling DNA damage in the absence of PARP1. Unfortunately, we have similarly been unable to disrupt the two Dictyostelium SSB responsive ARTs (Adprt1b and Adprt2) in combination.…”

“…PARP1, the founding member of the ART family, is recruited to single strand breaks (SSBs), becomes activated, and modifies a number of proteins at DNA lesions, including itself (Caldecott, 2008;Krishnakumar and Kraus, 2010). Resolution of SSBs is reduced in the presence of ART inhibitors (ARTi), or following depletion of PARP1 (Ding et al, 1992;Durkacz et al, 1980;Fisher et al, 2007;Morgan and Cleaver, 1983;Schraufstatter et al, 1986). Similarly, mice disrupted in the parp1 gene are sensitive to DNA damaging agents that induce base damage or strand breakage and exhibit delayed kinetics of SSB repair (SSBR) (de Murcia et al, 1997; Le Page et al, 2003;Masutani et al, 1999;Trucco et al, 1998;Trucco et al, 1999).…”

Radiation-induced mitotic catastrophe in PARG-deficient cells

“…Similarly, a loss-of-function mutation in the Drosophila melanogaster PARG gene led to increased lethality associated with PAR-accumulation-induced neurodegeneration at the larval stages (Hanai et al, 2004). Attempts to transiently silence PARG expression with siRNA led to controversial results: either protection (Blenn et al, 2006) or sensitivity (Fisher et al, 2007) towards H 2 O 2 -induced cell death, which might be explained by differences in cell types analyzed (HeLa versus A549 cells).…”