( E) Both DNA- and C-terminal domains contributed to PARP-1 protein dynamic binding to chromatin in vivo. All isoforms containing the Cat domain restored pADPr accumulation. PARP-1 deletional isoforms were expressed in the parg 27.1 parp-1 C03256 mutant flies. ( D) PARP-1 deletional isoform activity assay in vivo. All isoforms without ZI and ZII localized in active open chromatin only, resulting in the separation of red and green in the overlay. All isoforms of PARP-1 carrying ZI and ZII demonstrate colocalization with DNA resulting in the yellow color in the overlay. Green is the fluorescence of proteins fused to YFP, red is DNA. ( C) Localization of deletional recombinant isoforms of PARP-1 in salivary gland polytene chromosomes. ( B) Structure of recombinant-transgenic PARP-1 constructs for in vivo experiments. The red arrow indicates automodification of PARP-1. The dotted blue line indicates known interactions between the domains induced by interactions with damaged DNA ( 21). The PARP signature (PS) is an evolutionarily conserved PARP-1 catalytic site in the Cat domain. ( A) Domains of PARP-1: The N-terminal DNA-binding domain (DBD) containing Zn fingers: ZI, ZII, ZIII (ZF domains 1, 2, and 3), the automodification domain (A), the only domain of PARP-1 known to accept pADPr, the WGR domain (W), and the C-terminal catalytic domain (C). N- and C-terminal PARP-1 domains contribute to PARP-1 protein localization genome-wide in vivo. The presence of DBD and C-terminal domains is required for PARP-1-dependent chromatin condensation in vitro ( 34).įig. Binding of PARP-1 to histones has been shown to be regulated by its C-terminal subdomains ( 15, 16, 35). Without the DBD, PARP-1 cannot bind to or be activated by DNA ( 15, 34). The N-terminal DBD and the C-terminal ZIII-A-W-Cat domains are responsible for PARP-1 interaction with chromatin ( 15, 34).
An automodified PARP-1 loses its ability to interact with DNA ( 21, 31, 32) and serves as a “shuttle” for proteins of chromatin ( 33). The A domain of PARP-1 is the primary target of PARP-1 activity and becomes automodified by pADPr upon PARP-1 activation ( 10). During the DNA damage response, ZI, ZIII, W, and Cat domains of PARP-1 form a stable active complex around a fragment of broken DNA in vitro ( Fig. The third ZIII represents a protein interaction subdomain and mediates interprotein interactions of other PARP-1 domains ( 29, 30). The DNA-binding domain contains three Zinc fingers, ZI, ZII, and ZIII, of which only ZI and ZII are capable of interacting with DNA ( 10, 26– 28). PARP-1 consists of three core domains: the N-terminal DNA binding domain (DBD), the middle automodification domain (A), and the C-terminal catalytic domain (C) ( Fig. Cooperation between the DBD and C-terminal domain occurs in response to heat shock (HS), allowing PARP-1 to scan chromatin for specific binding sites. This long-term activation of PARP-1 results in a continuous accumulation of pADPr, which maintains chromatin in the loosened state around a certain locus so that the transcription machinery has continuous access to DNA. The long-term chromatin loosening required to sustain transcription takes place when the C-terminal domain of PARP-1 binds to chromatin by interacting with histone H4 in the nucleosome. This “hit and run” activation of PARP-1 initiates the DNA repair pathway at a specific point. Interaction between the DBD and damaged DNA leads to a short-term binding and activation of PARP-1. Using multiple deletional isoforms of PARP-1, lacking one or another of its three domains, as well as consisting of only one of those domains, we demonstrate that different functions of PARP-1 are coordinated by interactions among these domains and their targets. The mechanisms coordinating the functions of these domains and determining the positioning of PARP-1 in chromatin remain unknown. PARP-1 consists of three functional domains: the N-terminal DNA-binding domain (DBD) containing three zinc fingers, the automodification domain (A), and the C-terminal domain, which includes the protein interacting WGR domain (W) and the catalytic (Cat) subdomain responsible for the poly(ADP ribosyl)ating reaction. Poly(ADP-ribose) polymerase 1 (PARP-1) is a multidomain multifunctional nuclear enzyme involved in the regulation of the chromatin structure and transcription.
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