Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins.

Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribose) polymerases (PARPs). PARP-1, the best understood and until recently the only known member of this family, is a DNA damage signal protein catalyzing its automodification with multiple, variably sized ADP-ribose polymers that may contain up to 200 residues and several branching points. Through these polymers, PARP-1 can interact noncovalently with other proteins and alter their functions. Here we report the discovery of a poly(ADP-ribose)-binding sequence motif in several important DNA damage checkpoint proteins. The 20-amino acid motif contains two conserved regions: (i) a cluster rich in basic amino acids and (ii) a pattern of hydrophobic amino acids interspersed with basic residues. Using a combination of alanine scanning, polymer blot analysis, and photoaffinity labeling, we have identified poly(ADP-ribose)-binding sites in the following proteins: p53, p21(CIP1/WAF1), xeroderma pigmentosum group A complementing protein, MSH6, DNA ligase III, XRCC1, DNA polymerase epsilon, DNA-PK(CS), Ku70, NF-kappaB, inducible nitric-oxide synthase, caspase-activated DNase, and telomerase. The poly(ADP-ribose)-binding motif was found to overlap with five important functional domains responsible for (i) protein-protein interactions, (ii) DNA binding, (iii) nuclear localization, (iv) nuclear export, and (v) protein degradation. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions.

Poly ADP-ribosylation: a DNA break signal mechanism.

Recent evidence obtained with transgenic knockout mice suggests that the enzyme poly(ADP-ribose)polymerase (PARP) does not play a direct role in DNA break processing. Nevertheless, inactivation of the catalytic or the DNA nick-binding functions of PARP affects cellular responses to genotoxins at the level of cell survival, sister chromatid exchanges and apoptosis. In the present report, we conceptualize the idea that PARP is part of a DNA break signal mechanism. In vitro screening studies revealed the existence of a protein family containing a polymer-binding motif of about 22 amino acids. This motif is present in p53 protein as well as in MARCKS, a protein involved in the regulation of the actin cytoskeleton. Biochemical analyses showed that these sequences are directly targeted by PARP-associated polymers in vitro, and this alters several molecular functions of p53- and MARCKS protein. PARP-deficient knockout mice from transgenic mice were found to exhibit several phenotypic features compatible with altered DNA damage signaling, such as downregulation and lack of responsiveness of p53 protein to genotoxins, and morphological changes compatible with MARCKS-related cytoskeletal dysfunction. The knockout phenotype could be rescued by stable expression of the PARP gene. We propose that PARP-associated polymers may recruit signal proteins to sites of DNA breakage and reprogram their functions.

Poly(ADP-ribose) modulates the properties of MARCKS proteins.

In mammalian cells, the formation of DNA strand breaks is accompanied by synthesis of poly(ADP-ribose). This nucleic acid-like homopolymer may modulate protein functions by covalent and/or noncovalent interactions. Here we show that poly(ADP-ribose) binds strongly to the proteins of the myristoylated alanine-rich C kinase substrate (MARCKS) family, MARCKS and MARCKS-related protein (also MacMARCKS or F52). MARCKS proteins are myristoylated proteins associated with membranes and the actin cytoskeleton. As targets for both protein kinase C (PKC) and calmodulin (CaM), MARCKS proteins are thought to mediate cross-talk between these two signal transduction pathways. Dot blot assays show that poly(ADP-ribose) binds to MARCKS proteins at the highly basic effector domain. Complex formation between MARCKS-related protein and CaM as well as phosphorylation of MARCKS-related protein by the catalytic subunit of PKC are strongly inhibited by equimolar amounts of poly(ADP-ribose), suggesting a high affinity of poly(ADP-ribose) for MARCKS-related protein. Binding of MARCKS-related protein to membranes is also inhibited by poly(ADP-ribose). Finally, poly(ADP-ribose) efficiently reverses the actin-filament bundling activity of a peptide corresponding to the effector domain and inhibits the formation of actin filaments in vitro. Our results suggest that MARCKS proteins and actin could be targets of the poly(ADP-ribose) DNA damage signal pathway.

Poly(ADP-ribose) binds to specific domains of p53 and alters its DNA binding functions.

DNA strand breaks are potential interaction sites for the nuclear enzyme poly(ADP-ribose) polymerase (PARP; E.C. 2.4.2.30) and the tumor suppressor protein p53. Both proteins bind and respond to DNA breaks and both play a role in DNA damage signaling. A temporary colocalization and complex formation between these proteins has been demonstrated in mammalian cells. Here we show that free and poly(ADP-ribose) polymerase-bound ADP-ribose polymers target three domains in p53 protein for strong noncovalent interactions. The polymer binding sites could be mapped to two amino acid sequences in the sequence-specific core DNA binding domain of p53 (amino acid positions 153-178 and 231-253) and another one in the oligomerization domain (amino acids 326-348). In mobility shift experiments, poly(ADP-ribose) effectively prevented and reversed p53 binding to the palindromic p53 consensus sequence. Additionally, poly(ADP-ribose) also interfered with the DNA single strand end binding of p53. The results suggest that ADP-ribose polymers could play a role in regulating the DNA binding properties of p53.