PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage.

Poly(ADP-ribose) polymerase-2 (PARP-2) is one of three human PARP enzymes that are potently activated during the cellular DNA damage response (DDR). DDR-PARPs detect DNA strand breaks, leading to a dramatic increase in their catalytic production of the posttranslational modification poly(ADP-ribose) (PAR) to facilitate repair. There are limited biochemical and structural insights into the functional domains of PARP-2, which has restricted our understanding of how PARP-2 is specialized toward specific repair pathways. PARP-2 has a modular architecture composed of a C-terminal catalytic domain (CAT), a central Trp-Gly-Arg (WGR) domain and an N-terminal region (NTR). Although the NTR is generally considered the key DNA-binding domain of PARP-2, we report here that all three domains of PARP-2 collectively contribute to interaction with DNA damage. Biophysical, structural and biochemical analyses indicate that the NTR is natively disordered, and is only required for activation on specific types of DNA damage. Interestingly, the NTR is not essential for PARP-2 localization to sites of DNA damage. Rather, the WGR and CAT domains function together to recruit PARP-2 to sites of DNA breaks. Our study differentiates the functions of PARP-2 domains from those of PARP-1, the other major DDR-PARP, and highlights the specialization of the multi-domain architectures of DDR-PARPs

PARP-2 and PARP-3 are selectively activated by 5\u27 phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1.

PARP-1, PARP-2 and PARP-3 are DNA-dependent PARPs that localize to DNA damage, synthesize poly(ADP-ribose) (PAR) covalently attached to target proteins including themselves, and thereby recruit repair factors to DNA breaks to increase repair efficiency. PARP-1, PARP-2 and PARP-3 have in common two C-terminal domains-Trp-Gly-Arg (WGR) and catalytic (CAT). In contrast, the N-terminal region (NTR) of PARP-1 is over 500 residues and includes four regulatory domains, whereas PARP-2 and PARP-3 have smaller NTRs (70 and 40 residues, respectively) of unknown structural composition and function. Here, we show that PARP-2 and PARP-3 are preferentially activated by DNA breaks harboring a 5\u27 phosphate (5\u27P), suggesting selective activation in response to specific DNA repair intermediates, in particular structures that are competent for DNA ligation. In contrast to PARP-1, the NTRs of PARP-2 and PARP-3 are not strictly required for DNA binding or for DNA-dependent activation. Rather, the WGR domain is the central regulatory domain of PARP-2 and PARP-3. Finally, PARP-1, PARP-2 and PARP-3 share an allosteric regulatory mechanism of DNA-dependent catalytic activation through a local destabilization of the CAT. Collectively, our study provides new insights into the specialization of the DNA-dependent PARPs and their specific roles in DNA repair pathways

The Antimicrobial Efficacy of Electroless Plated Copper and Gold Nylon

The work presented in this thesis is a quantitative and qualitative assessment of electroless plated copper and gold nylon fabrics for the potential use as effective antimicrobial agents. Tests included, but were not limited to Kirby-Bauer Disk-Diffusion testing, metallic release studies, surface resistivity testing and Scanning Electron Microscope evaluations. The Kirby-Bauer testing took place over the course of a seven-day period using Staphylococcus epidermidis, Methicillin-Resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Zones of inhibition were recorded at each 24-hour interval up to seven days or a time when bacteria growth was visibly present on the plated fabric. Gold and copper plated fabric inhibited most of the tested bacteria for seven days. The results of these studies indicate that copper and gold have the potential to be used as broad-spectrum antimicrobials and an effective alternative to other antimicrobials. Additionally, metal release studies were conducted using Atomic Absorption spectroscopy. Copper release into normal saline (0.9% w/v), tryptic soy broth (TSB), and nanopure water confirmed that variable quantities of copper were present throughout the seven days of analysis. The concentration of copper released into the various media ranged from approximately 110 parts per million (ppm), released into TSB within the first hour, to approximately 0.7 ppm released into nanopure water by the seventh day of the study. Scanning Electron Microscope (SEM) images indicated possible hydrogen brittlement or expedited plating had taken place, but copper was present on the fabric in measureable quantities. Subsequent to this analysis, it is probable that copper and/or gold plated fabrics will be of practical application and an effective alternative to other exhausted, microorganism-resistant antimicrobials in current application

Poly(ADP-ribose) polymerase 2 (PARP-2) mechanism of DNA damage recognition and allosteric activation

Poly(ADP-ribose), or PAR, is a transient, posttranslational modification catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes. PARPs utilize NAD+ as a substrate to generate PAR for self-attachment (termed automodification) or for attachment to target proteins. PARPs have been implicated in processes including, but not limited to: DNA damage repair, metabolic regulation, and programmed cellular death. There are 17 members of the PARP family of enzymes each characterized by the highly homologous C-terminal ADP-ribose transferase fold (ART) of the CAT domain. In contrast to the homology in this domain, the regulatory domains, such as N-terminal region (NTR) and tryptophan(W)-glycine(G)-arginine(R) (WGR) domain, are much less explored. The lack of identified structural information about PARP regulatory domains leaves a substantial gap in our knowledge. PARPs 1, 2, and 3 exhibit DNA damage-dependent activation and are known as DNA damage response PARPS (DDR-PARPs). During the response to DNA damage, DDR-PARPs recognize DNA breaks and increase the production of PAR. PAR aids in the recruitment of subsequent repair factors to the site of DNA damage. Together, PARP-1 and PARP-2 are essential enzymes, both playing key roles in the DNA damage response, but also in other cellular programs such as gene transcription. Despite extensive characterization of PARP-1, there is limited biochemical and structural analysis of PARP-2, which has a unique domain structure and several distinct cellular roles. Prior to the work presented here, several key aspects of PARP-2 mechanism were not established and thus limited our understanding of PARP-2 function, such as how PARP-2 selectively recognizes DNA repair intermediates and acts within a specific repair pathway. To clarify the role of PARP-2 in DNA repair pathways and the DNA damage response, we have undertaken a structural, biochemical, and cellular investigation of PARP-2. The work presented here has resulted in several novel insights into PARP-2 structure and function. Specifically, we conclude that PARP-2 is selectively activated on 5’phosphorylated DNA breaks, which implicates PARP-2 specific activation just prior to DNA break ligation in the DNA repair process. The data establishes that the NTR is natively disordered, recognizes specific DNA breaks, and requires assembly with other PARP-2 domains for a functional DNA damage recognition response. As a result of this research, it is now known that PARP-2 acts through an allosteric mechanism similar to PARP-1, whereby DNA damage recognition is transmitted to the catalytic domain (CAT) through interdomain communication. Additionally, it is now appreciated that the enzymatic activity of PARP-2 is regulated through the autoinhibitory helical domain (HD), a subdomain of CAT, which locally unfolds upon activation. The unfolding of a region in the HD is a unique allosteric mechanism by which the robust catalytic potential of PARP-2 is tightly regulated. Our comprehensive biochemical and structural approach to study PARP-2 in DNA damage repair further differentiates PARP-2 from other DNA damage-dependent PARPs and leads to a more detailed understanding of the activation mechanism of DDR-PARPs. PARP inhibition is a novel, molecular targeted approach that specifically kills certain cancers with DNA repair deficiencies such as BRCA-1/2 deficient breast and ovarian cancer. This single agent approach, termed “synthetic lethality”, has advanced into several clinical trials leading to the first approved PARP inhibitor, Olaparib (Lynparza™), for advanced ovarian cancers with genetically abnormal BRCA status. Current PARP inhibitors, including Olaparib, bind to the NAD+ binding site that is well conserved across PARPs, and therefore inhibit both PARP-1 and PARP-2 similarly. Overall, this work has presented a foundation to the understanding of DNA damage recognition and activation of PARP-2, having implications for a better understanding of DDR-PARP biology, which could ultimately lead to improved therapeutic options

Ubiquitin stimulated reversal of topoisomerase 2 DNA-protein crosslinks by TDP2

Intramural research program of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS) [1Z01ES102765 to R.S.W.]; Work in the F.C.L. lab is supported by Ministerio de Economía y Competitividad, Gobierno de España [SAF2017-89619-R, European Regional Development Fund]; European Research Council [ERC-CoG-2014-647359]; University of Seville Predoctoral Studentship [PIF-2011 to J.A.L.]; M.J.S. is supported by Mayo Clinic start-up funds and the Center for Biomedical Discovery new investigator funds; Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory; Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [W-31-109-Eng-38]; SAXS data were collected at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported by DOE Office of Biological and Environmental Research. Additional support comes from the National Institute of Health project ALS-ENABLE [P30 GM124169]; High-End Instrumentation Grant [S10OD018483]. Funding for open access charge: US government, Intramural NIH.Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA-protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface.S

Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1

Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins