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.
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.
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