Crystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNA interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks

“…In contrast with structural analysis of the separated domains 22 , we show that DNA binding by both zinc-finger domains is essential to damage recruitment in vivo, and that ZnF1 and ZnF2 domains from separate PARP1 molecules act as a functional unit to generate a dimeric binding module that specifically recognizes the single-strand / double-strand transition at a recessed DNA break. Mutational analysis in vitro and in cells demonstrates the functional requirement for zinc-finger dimerisation and reveals a mechanism for bringing two PARP1 molecules into close proximity at a DNA break as a prerequisite for transmodification.…”

“…Significantly, the major differences observed map to the loop containing Met 43 and Phe 44, which interacts with the faces of the terminal base-pair in the context of the isolated ZnF1, but forms the protein-protein interface with ZnF2 in the context of the intact PARP1-DBD. If ZnF1 has a role of mediating nonspecific PARP1 interaction with undamaged DNA, as has been suggested 21,22 , then this may be mediated by Phe 44 and the 2-3 loop of ZnF1, independently of ZnF2. However our in vivo data show unambiguously that recruitment of PARP1 to damaged DNA is critically dependent on the ability of both ZnF domains to interact with DNA, and with each other.…”

“…While this work was in progress, structures of the separate PARP1 ZnF domains bound to blunt-ended DNA oligonucleotides were reported 22 . The DNA interactions made by ZnF2 in our study closely resembles those of the isolated domain, however the behavior of ZnF1 is radically different (see DISCUSSION).…”

“…Structures of the isolated PARP1 ZnF1 and ZnF2 domains have been determined in complex with blunt-ended DNA oligonucleotides 22 . The structure of the isolated ZnF2-DNA complex is consistent with our observation that ZnF2 in the intact PARP1-DBD complex can only bind to duplex DNA at strand discontinuities, and with previous work suggesting that ZnF2 plays the major role in gap-recognition 23,35 .…”

“…However, despite recent work [20][21][22] the biochemical and structural mechanism by which PARP1 recognises and is activated by DNA gaps and breaks remains obscure. A substantial literature suggests that PARP1 activation involves dimerisation mediated by N-terminal and central domains of the protein, with consequent automodification occurring in trans as an intermolecular reaction 1,7,[23][24][25][26] .…”