Stimulation of the DNA-dependent Protein Kinase by Poly(ADP-Ribose) Polymerase

Abstract

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD ؉ in a reaction that is blocked by the PARP inhibitor 1,5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.Biochemical pathways that function in the recognition and repair of DNA damage are critical for maintaining genomic integrity. Potentially devastating DNA damage in the form of double-strand breaks (DSBs) 1 can occur when cells are exposed to ionizing radiation, oxidative stress and radiomimetic drugs. The DNA-dependent protein kinase (DNA-PK) is a key component of DNA DSB rejoining pathways in mammalian cells. DNA-PK is a heterotrimeric enzyme complex comprised of a 460-kDa catalytic subunit (1) and a regulatory component consisting of the Ku70 and Ku80 proteins (2, 3). The Ku70 and Ku80 proteins form a heterodimeric complex that binds to the ends of double-stranded DNA with high affinity (4 -8). The catalytic subunit of DNA-PK (DNA-PKcs) binds to the Ku70/80 complex in the presence of double-stranded DNA (9) and phosphorylates a wide variety of protein substrates in vitro on serine and threonine residues (10, 11). The protein kinase activity of DNA-PK is autoregulatory; in the absence of a phosphorylation substrate, DNA-PKcs autophosphorylates and dissociates from the Ku70/80-DNA complex (12).Evidence that DNA-PK plays an integral role in the repair of DNA DSB has been provided through the characterization of rodent cell lines that have mutations that disrupt the expression of the Ku80 (13-19) or DNA-PKcs (20 -24). Although it is clear that DNA-PK is an important component of mammalian DNA DSB repair pathways, it is not known how the enzyme participates in these processes. In vitro, DNA-PK preferentially phosphorylates protein substrates that co-localize on the same DNA molecule (3,25). This suggests that the specificity of the phosphorylation reaction may be regulated, in part, via the co-localization of the enzyme and substrate target on DNA. Based on these data, it has been proposed that DNA-PK could participate in the DNA rejoining reaction by phosphorylating DNA repair factors that co-localize with it on broken DNA en...

Poly(ADP-ribose) polymerase-1 polymorphisms, expression and activity in selected human tumour cell lines

“…The assay we used in this study, by the use of an oligonucleotide, measures total stimulatable PARP activity and therefore cellular events that might activate PARP-1 endogenously, such as oxidative DNA damage, will not contribute to the variation in activity observed. There are numerous factors that might have an impact on PARP-1 activity; DNA-PK may activate PARP-1 by phosphorylation (Ruscetti et al, 1998) or suppress PARP activity through sequestration of DNA ends that serve as an important stimulator for both enzymes (Ariumi et al, 1999). Inactive DNA-PK has also been shown to suppress the activity of PARP-1, an effect that was not due to substrate competition, as DNA ends were provided in excess (Veuger et al, 2004).…”

Systems analysis of the prostate tumor suppressor NKX3.1 supports roles in DNA repair and luminal cell differentiation

“…Such phosphorylation was proposed to lead to DNA damage-dependent changes in their transcriptional activities. ADP-ribosylation mediated by PARP1 can stimulate the ability of DNA-PK to phosphorylate protein substrates 40 . Our interactome data provide a possible mechanism underlying the previously observed localization of NKX3.1 to sites of DNA damage 24 , although the functional consequences of these interactions for NKX3.1 transcriptional activity remain to be established.…”

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

“…Many studies have linked PARP-1 and Ku either directly or as components of multiprotein complexes (Ruscetti et al, 1998;Ariumi et al, 1999;Galande and Kohwi-Shigematsu, 1999;Grote et al, 2006;Yin and Glass, 2006;Hossain et al, 2009). Although cell lines' extraction procedures and coimmunoprecipitation protocols vary between studies, there have been consistent observations of Ku/PARP-1 interactions and a frequent observation that PARP activity or DNA-PK mediated phosphorylation modulates the complexes.…”

“…Of note, these earlier experiments were performed with naı¨ve factors or extracts that were prepared from cells that had not been exposed to a DNA damaging agent such as the bleomycin. In addition, in at least one study (Ruscetti et al, 1998), Ku/PARP-1 binding was determined to be resistant to DNase I digestion, which would disrupt colocalization of two factors to the same DNA molecule or DNA-dependent bridging interactions between the two factors. However, unlike the ethidium bromide used in our study, protein-DNA interactions can be resistant to DNAse I.…”

“…Genetic studies have established a role for PARP-1 in HR (Hochegger et al, 2006;Idogawa et al, 2007), whereas biochemical and genetic studies have linked PARP-1 to NHEJ (Schultz et al, 2003;Perrault et al, 2004;Audebert et al, 2006;Wang et al, 2006). PARP-1 has been shown to interact with the NHEJ proteins, Ku antigen and DNA-dependent protein kinase (DNA-PK) with reciprocal modifications and mutual alterations in activity suggested (Ruscetti et al, 1998;Ariumi et al, 1999;Galande and KohwiShigematsu, 1999). Another study indicated that PARP-1 may specifically facilitate HR by decreasing HR-inhibitory effects of Ku antigen (Hochegger et al, 2006).…”