The Zn3 Domain of Human Poly(ADP-ribose) Polymerase-1 (PARP-1) Functions in Both DNA-dependent Poly(ADP-ribose) Synthesis Activity and Chromatin Compaction

Abstract

PARP-1 is involved in multiple cellular processes, including transcription, DNA repair, and apoptosis. PARP-1 attaches ADP-ribose units to target proteins, including itself as a posttranslational modification that can change the biochemical properties of target proteins and mediate recruitment of proteins to sites of poly(ADP-ribose) synthesis. Independent of its catalytic activity, PARP-1 binds to chromatin and promotes compaction affecting RNA polymerase II transcription. PARP-1 has a modular structure composed of six independent domains. Two homologous zinc fingers, Zn1 and Zn2, form the DNAbinding module. Zn1-Zn2 binding to DNA breaks triggers catalytic activity. Recently, we have identified a third zinc binding domain in PARP-1, the Zn3 domain, which is essential for DNAdependent PARP-1 activity. The crystal structure of the Zn3 domain revealed a novel zinc-ribbon fold and a homodimeric Zn3 structure that formed in the crystal lattice. Structureguided mutagenesis was used here to investigate the roles of these two features of the Zn3 domain. Our results indicate that the zinc-ribbon fold of the Zn3 domain mediates an interdomain contact crucial to assembly of the DNA-activated conformation of PARP-1. In contrast, residues located at the Zn3 dimer interface are not required for DNA-dependent activation but rather make important contributions to the chromatin compaction activity of PARP-1. Thus, the Zn3 domain has dual roles in regulating the functions of PARP-1. Poly(ADP-ribose) polymerase-1 (PARP-1)4 is a multifunctional enzyme that covalently attaches ADP-ribose to target proteins (1, 2). Using NAD ϩ as a donor, PARP-1 can polymerize multiple units of ADP-ribose to create long and branched polymers termed poly(ADP-ribose) (PAR). This post-translational modification can change the biochemical properties and cellular location of acceptor proteins. The main substrate of PARP-1 in the cell is PARP-1 itself (automodification activity), although PARP-1 also ADP-ribosylates various substrates, including core histones, histone H1, and transcription regulatory proteins (3-7). PARP-1 can bind to nucleosomes and induce chromatin compaction, an activity that is reversed by automodification of PARP-1 (8, 9). Ecdysone-and heat shockinducible "puffing" (i.e. chromatin decondensation) of specific loci in Drosophila requires PARP-1 enzymatic activity and is accompanied by the accumulation of PAR in the puffs (10). In vivo PARP-1 localizes to actively transcribed RNA polymerase II promoters (11).PARP-1 also plays important roles in DNA repair. Following DNA damage, PARP-1 is recruited to sites of DNA lesion by its ability to bind DNA strand breaks. Binding to DNA strand breaks stimulates the automodification activity of PARP-1 and could lead to recruitment of DNA repair machinery to the sites of damage through an interaction with XRCC1 (12, 13). More recent results support an alternative model whereby PARP-1 acts to protect DNA single strand breaks from being converted to more deleterious double strand breaks, until they ca...

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

“…However, in the presence of a hairpin DNA molecule with a 'damaged' 3'-recessed end we observed robust co-precipitation of PARP1 1-372 and PARP1-DBD by GST-PARP1-DBD and by GST-ZnF1 and GST-ZnF2. We did not observe any dimeric interactions, in the presence or absence of DNA, involving ZnBD3, which had been previously suggested to mediate PARP1 dimerisation 29 .…”

“…The DNA damage-dependent dimerization of the PARP1-DBD we describe here is precisely analogous and fulfills the essential requirement of bringing two PARP1 molecules into close proximity, to allow trans-ADP-ribosylation. This is likely to be accompanied by substantial conformational rearrangements of the whole molecule, involving intra-and inter-molecular cooperation between other PARP1 domains (see for example 29 ) that ultimately enable the catalytic domain of one molecule to access and ADP-ribosylate the PAR-acceptor sites on the other. Elucidation of the detailed interactions and conformational states that facilitate specific trans-automodification of PARP1 and subsequent hetero-modification of down-stream targets remains a significant challenge.…”

Regulation of Poly(ADP-ribose) Polymerase-1-dependent Gene Expression through Promoter-directed Recruitment of a Nuclear NAD+ Synthase

“…To explore the NAD ϩ -dependent activity of PARP-1 in the control of transcription, we used a variety of biochemical, molecular, cell-based, and genomic assays. We have shown previously that PARP-1 in the absence of NAD ϩ represses ER␣-dependent transcription in an in vitro transcription assay with chromatin templates (22,23,43). The repression by PARP-1 is relieved following addition of NAD ϩ and subsequent automodification of PARP-1 (22,23,43).…”

“…We have shown previously that PARP-1 in the absence of NAD ϩ represses ER␣-dependent transcription in an in vitro transcription assay with chromatin templates (22,23,43). The repression by PARP-1 is relieved following addition of NAD ϩ and subsequent automodification of PARP-1 (22,23,43). Using a similar in vitro transcription assay with chromatin templates, we examined whether the nuclear NAD ϩ synthase NMNAT-1 could produce NAD ϩ to support the activity of PARP-1 and relieve the PARP-1-mediated repression in these assays.…”

Acetyl-L-Carnitine and Nicotinamide for Prevention of Type 1 Diabetes. I-Literature Review which Gave Support to the Treatment. II-Case Report, Evaluation of Five Years Treatment

“…Nicotinamide was a potent inhibitor of pro-inflammatory cytokines through inhibition of PARP [70,71], and this activity was not only based on its anti-apoptotic and anti-necrotic action due to PARP gene inhibition but also on its capacity to be a NAD + substrate [64,67]. With an enzyme Km for DNA of 86.5 µM, and an apparent Km for NAD + of 20 µM [70,[72][73][74], a nicotinamide dose in humans of 25 mg/kg body weight reached a plasma concentration of 0.3 mM [64] demonstrating that nicotinamide at this dose was sufficient for PARP inhibition [70].…”