PARP-1 cleaves NAD+ and transfers the resulting ADP-ribose moiety onto target proteins and onto subsequent polymers of ADP-ribose. An allosteric network connects PARP-1 multi-domain detection of DNA damage to catalytic domain structural changes that relieve catalytic autoinhibition; however, the mechanism of autoinhibition is undefined. Here, we show using the non-hydrolyzable NAD+ analog benzamide adenine dinucleotide (BAD) that PARP-1 autoinhibition results from a selective block on NAD+ binding. Following DNA damage detection, BAD binding to the catalytic domain leads to changes in PARP-1 dynamics at distant DNA-binding surfaces, resulting in increased affinity for DNA damage, and providing direct evidence of reverse allostery. Our findings reveal a two-step mechanism to activate and to then stabilize PARP-1 on a DNA break, indicate that PARP-1 allostery influences persistence on DNA damage, and have important implications for PARP inhibitors that engage the NAD+ binding site.

The success of poly(ADP-ribose) polymerase–1 (PARP-1) inhibitors (PARPi) to treat cancer relates to their ability to trap PARP-1 at the site of a DNA break. Although different forms of PARPi all target the catalytic center of the enzyme, they have variable abilities to trap PARP-1. We found that several structurally distinct PARPi drive PARP-1 allostery to promote release from a DNA break. Other inhibitors drive allostery to retain PARP-1 on a DNA break. Further, we generated a new PARPi compound, converting an allosteric pro-release compound to a pro-retention compound and increasing its ability to kill cancer cells. These developments are pertinent to clinical applications where PARP-1 trapping is either desirable or undesirable.

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNAscanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA. In particular, the zinc finger 1 (ZF1) of Egr-1 in the nonspecific complex is mainly dissociated from DNA and undergoes collective motions on a nanosecond timescale, whereas zinc fingers 2 and 3 (ZF2 and ZF3, respectively) are bound to DNA. This was totally unexpected because the previous crystallographic studies of the specific complex indicated that all of Egr-1's three zinc fingers are equally involved in binding to a target DNA site. Mutations that are expected to enhance ZF1's interactions with DNA and with ZF2 were found to reduce ZF1's domain motions in the nonspecific complex suggesting that these interactions dictate the dynamic behavior of ZF1. By experiment and computation, we have also investigated kinetics of Egr-1's translocation between two nonspecific DNA duplexes. Our data on the wild type and mutant proteins suggest that the domain dynamics facilitate Egr-1's intersegment transfer that involves transient bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger domains for Egr-1 to scan DNA efficiently in the nucleus.NMR spectroscopy | target search process | interdomain dynamics | protein-DNA interactions | simulation I n cellular responses to various stimuli such as signals and stresses, gene regulation by transcription factors is of fundamental importance. Egr-1 (also known as Zif268) is an inducible transcription factor with crucial roles particularly in the brain and cardiovascular systems in mammals. In the brain, Egr-1 is induced by synaptic signals in an activity-dependent manner and activates genes for long-term memory formation and consolidation (1, 2). In the cardiovascular system, Egr-1 is a stress-inducible transcription factor that activates the genes for initiating defense responses against vascular stress and injury (3, 4). Given the short lifetime of induced Egr-1 (typically ∼2 h) (3), rapid gene activation by Egr-1 is important in these biological processes that require an immediate response to the stimuli.The induced Egr-1 protein has to initiate its role by searching for its target DNA sites among billions of DNA base pairs in the nucleus. In the DNA scanning process, transcription factors need to discriminate their target sites from nonspecific sites based on relatively minor differences in DNA structure and sequence. Crystallographic studies demonstrated that Egr-1 recognizes its 9-bp target sequence, GCGTGGGCG, as a monomer via zinc finger domains 1, 2, and 3 (hereafter referred to as ZF1, ZF2, and ZF3) that contact 3 ...

Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNAscanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering.protein-DNA interactions | DNA scanning | target search | kinetics | dynamics A rtificial transcription factors and DNA-modifying enzymes have gained popularity as powerful means for artificial gene regulation and genome editing (1-7). Successful applications were reported on artificial zinc-finger (ZF) proteins engineered to exhibit a desired sequence specificity in binding to DNA (1-5). Artificial ZF transcription factors, comprising engineered ZFs and transactivation or repression domains, are used to regulate particular genes (1-3). ZF nucleases (ZFNs), comprising engineered ZFs and a FokI nuclease domain (ND), can site-specifically cleave DNA at particular sequences and allow for genome editing in vivo (4, 5). ZFN-based gene therapy for HIV infection is currently under phase 2 clinical trials, yielding some successful cases (8). Other studies suggested that ZF-based gene control could also be therapeutically effective for hemophilia (9) and Parkinson's disease (10). For the success of these technologies, however, two issues, toxicity and low efficiency, should be resolved (4, 5, 11). Regarding the latter issue, some studies (11)(12)(13)(14) suggest that, despite high affinities for target DNA, the artificial proteins do not necessa...

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the ‘arginine anchor' on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA.