Marie-France Langelier

Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1.

Dissecting DNA Repair Covalent modification of proteins can be a crucial regulatory event. Poly(ADP-ribose) polymerase 1 (PARP-1) (ADP, adenosine diphosphate) synthesizes poly(ADP-ribose), which is attached to and regulates proteins involved in DNA repair. Langelier et al. (p. 728; see the Perspective by Gagné et al.) use x-ray crystallography and biochemical analysis to demonstrate how PARP-1 detects DNA damage and how the interaction with DNA is coupled to poly(ADP-ribosyl)ation activity. An enzyme that binds to damaged DNA undergoes a structural reorganization that enhances its catalytic activity. Poly(ADP-ribose) polymerase–1 (PARP-1) (ADP, adenosine diphosphate) has a modular domain architecture that couples DNA damage detection to poly(ADP-ribosyl)ation activity through a poorly understood mechanism. Here, we report the crystal structure of a DNA double-strand break in complex with human PARP-1 domains essential for activation (Zn1, Zn3, WGR-CAT). PARP-1 engages DNA as a monomer, and the interaction with DNA damage organizes PARP-1 domains into a collapsed conformation that can explain the strong preference for automodification. The Zn1, Zn3, and WGR domains collectively bind to DNA, forming a network of interdomain contacts that links the DNA damage interface to the catalytic domain (CAT). The DNA damage–induced conformation of PARP-1 results in structural distortions that destabilize the CAT. Our results suggest that an increase in CAT protein dynamics underlies the DNA-dependent activation mechanism of PARP-1.

A Third Zinc-Binding Domain of Human Poly(ADP-ribose) Polymerase-1 Coordinates DNA-Dependent Enzyme Activation

Poly(ADP-ribose) polymerase-1 (PARP-1) is a chromatin-associated enzyme with multiple cellular functions, including DNA repair, transcriptional regulation, and cell signaling. PARP-1 has a modular architecture with six independent domains comprising the 113-kDa polypeptide. Two zinc finger domains at the N terminus of PARP-1 bind to DNA and thereby activate the catalytic domain situated at the C terminus of the enzyme. The tight coupling of DNA binding and catalytic activities is critical to the cellular regulation of PARP-1 function; however, the mechanism for coordinating these activities remains an unsolved problem. Here, we demonstrate using spectroscopic and crystallographic analysis that human PARP-1 has a third zinc-binding domain. Biochemical mutagenesis and deletion analysis indicate that this region mediates interdomain contacts important for DNA-dependent enzyme activation. The crystal structure of the third zinc-binding domain reveals a zinc ribbon fold and suggests conserved residues that could form interdomain contacts. The new zinc-binding domain self-associates in the crystal lattice to form a homodimer with a head-totail arrangement. The structure of the homodimer provides a scaffold for assembling the activated state of PARP-1 and suggests a mechanism for coupling the DNA binding and catalytic functions of PARP-1.

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.

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 post-translational 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 DNA-binding 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 DNA-dependent 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. Structure-guided 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.

Crystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA: STRUCTURAL AND FUNCTIONAL INSIGHTS INTO DNA-DEPENDENT PARP-1 ACTIVITY.

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.

PARP-1 mechanism for coupling DNA damage detection to poly(ADP-ribose) synthesis

Poly(ADP-ribose) polymerase 1 (PARP-1) regulates gene transcription, cell death signaling, and DNA repair through production of the posttranslational modification poly(ADP-ribose). During the cellular response to genotoxic stress PARP-1 rapidly associates with DNA damage, which robustly stimulates poly(ADP-ribose) production over a low basal level of PARP-1 activity. DNA damage-dependent PARP-1 activity is central to understanding PARP-1 biological function, but structural insights into the mechanisms underlying this mode of regulation have remained elusive, in part due to the highly modular six-domain architecture of PARP-1. Recent structural studies have illustrated how PARP-1 uses specialized zinc fingers to detect DNA breaks through sequence-independent interaction with exposed nucleotide bases, a common feature of damaged and abnormal DNA structures. The mechanism of coupling DNA damage detection to elevated poly(ADP-ribose) production has been elucidated based on a crystal structure of the essential domains of PARP-1 in complex with a DNA strand break. The multiple domains of PARP-1 collapse onto damaged DNA, forming a network of interdomain contacts that introduce destabilizing alterations in the catalytic domain leading to an enhanced rate of poly(ADP-ribose) production.

PARP-2 and PARP-3 are selectively activated by 5′ 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′ phosphate (5′P), 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.

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

ABSTRACT We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Photo-Cross-Linking of a Purified Preinitiation Complex Reveals Central Roles for the RNA Polymerase II Mobile Clamp and TFIIE in Initiation Mechanisms

ABSTRACT The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIEα closely approaches promoter DNA at nucleotide −10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIEα is obtained when the DNA template is artificially premelted in the −11/−1 region, suggesting that TFIIEα facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.

Structural Perspective on Mutations Affecting the Function of Multisubunit RNA Polymerases

SUMMARY High-resolution crystallographic structures of multisubunit RNA polymerases (RNAPs) have increased our understanding of transcriptional mechanisms. Based on a thorough review of the literature, we have compiled the mutations affecting the function of multisubunit RNA polymerases, many of which having been generated and studied prior to the publication of the first high-resolution structure, and highlighted the positions of the altered amino acids in the structures of both the prokaryotic and eukaryotic enzymes. The observations support many previous hypotheses on the transcriptional process, including the implication of the bridge helix and the trigger loop in the processivity of RNAP, the importance of contacts between the RNAP jaw-lobe module and the downstream DNA in the establishment of a transcription bubble and selection of the transcription start site, the destabilizing effects of ppGpp on the open promoter complex, and the link between RNAP processivity and termination. This study also revealed novel, remarkable features of the RNA polymerase catalytic mechanisms that will require additional investigation, including the putative roles of fork loop 2 in the establishment of a transcription bubble, the trigger loop in start site selection, and the uncharacterized funnel domain in RNAP processivity.

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

Summary 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.