Reversible post-translational modification by poly(ADP-ribose) (PAR) regulates chromatin structure, DNA repair and cell fate in response to genotoxic stress. PAR glycohydrolase (PARG) removes PAR ...chains from poly ADP-ribosylated proteins to restore protein function and release oligo(ADP-ribose) chains to signal damage. Here we report crystal structures of mammalian PARG and its complex with a substrate mimic that reveal an open substrate-binding site and a unique 'tyrosine clasp' enabling endoglycosidic cleavage of branched PAR chains.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Calcineurin phosphatase activity regulates the nuclear localization of the nuclear factor of activated T cells (NFAT) family of transcription factors during immune challenge. Calcineurin inhibitors, ...such as the cyclosporin A-cyclophilin A and FK506-FKBP12 complexes, regulate this enzymatic activity noncompetitively by binding at a site distinct from the enzyme active site. A family of endogenous protein inhibitors of calcineurin was recently identified and shown to block calcineurin-mediated NFAT nuclear localization and transcriptional activation. One such inhibitor, Down Syndrome Critical Region 1 (DSCR1), functions in T cell activation, cardiac hypertrophy, and angiogenesis. We have identified a small region of DSCR1 that is a potent inhibitor of calcineurin activity in vitro and in vivo.
The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential ...assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein–protein and protein–DNA interactions within NER complexes. We have investigated an essential protein–protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1‐XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell‐free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.
•Exo1 is a novel poly(ADP-ribose)-binding protein.•Poly(ADP-ribose)-binding of the N-terminal domain of Exo1 promotes its early recruitment to sites of DNA damage.•14-3-3 proteins restrain the damage ...association of Exo1 by suppressing its interactions with poly(ADP-ribose) and PCNA.•Poly(ADP-ribose) inhibits the 5′-to-3′ exonuclease activity and the 5′ flap endonuclease activity of Exo1.•Protein poly(ADP-ribosyl)ation is dispensable for the resection of DNA double-strand breaks.
Exonuclease 1 (Exo1) has important roles in DNA metabolic transactions that are essential for genome maintenance, telomere regulation and cancer suppression. However, the mechanisms for regulating Exo1 activity in these processes remain incompletely understood. Here, we report that Exo1 activity is regulated by a direct interaction with poly(ADP-ribose) (PAR), a prominent posttranslational modification at the sites of DNA damage. This PAR-binding activity promotes the early recruitment of Exo1 to sites of DNA damage, where it is retained through an interaction with PCNA, which interacts with the C-terminus of Exo1. The effects of both PAR and PCNA on Exo1 damage association are antagonized by the 14-3-3 adaptor proteins, which interact with the central domain of Exo1. Although PAR binding inhibits both the exonuclease activity and the 5′ flap endonuclease activity of purified Exo1, the pharmacological blockade of PAR synthesis does not overtly affect DNA double-strand break end resection in a cell free Xenopus egg extract. Thus, the counteracting effects of PAR on Exo1 recruitment and enzymatic activity may enable appropriate resection of DNA ends while preventing unscheduled or improper processing of DNA breaks in cells.
The NAD+-dependent protein deacetylase SIRT1 regulates energy metabolism, responses to stress, and aging by deacetylating many different proteins, including histones and transcription factors. The ...mechanisms controlling SIRT1 enzymatic activity are complex and incompletely characterized, yet essential for understanding how to develop therapeutics that target SIRT1. Here, we demonstrate that the N-terminal domain of SIRT1 (NTERM) can trans-activate deacetylation activity by physically interacting with endogenous SIRT1 and promoting its association with the deacetylation substrate NF-κB p65. Two motifs within the NTERM domain contribute to activation of SIRT1-dependent activities, and expression of one of these motifs in mice is sufficient to lower fasting glucose levels and improve glucose tolerance in a manner similar to overexpression of SIRT1. Our results provide insights into the regulation of SIRT1 activity and a rationale for pharmacological control of SIRT1-dependent activities.
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•The SIRT1 N-terminal domain regulates acetylation of NF-κB p65 and p53•NTERM physically interacts with SIRT1 and promotes binding to NF-κB p65•Two NTERM motifs interact with each other and contribute to these effects•One peptide motif lowers fasting glucose and improves glucose tolerance in vivo
The NAD+-dependent protein deacetylase SIRT1 regulates energy metabolism, responses to stress, and aging by deacetylating proteins, including histones and transcription factors. Ghisays et al. show that the catalytically inactive, N-terminal domain of SIRT1 regulates deacetylation activity in cells and improves glucose metabolism in mice, with implications for SIRT1-based therapeutics.
The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 Å crystal structure of AlkA complexed to DNA shows a large ...distortion in the bound DNA. The enzyme flips a 1‐azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1‐azaribose in the enzyme active site suggests an SN1‐type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin–helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.
DNA sliding clamps encircle DNA and provide binding sites for many DNA-processing enzymes. However, it is largely unknown how sliding clamps like proliferating cell nuclear antigen (PCNA) coordinate ...multistep DNA transactions. We have determined structures of
Sulfolobus solfataricus DNA ligase and heterotrimeric PCNA separately by X-ray diffraction and in complex by small-angle X-ray scattering (SAXS). Three distinct PCNA subunits assemble into a protein ring resembling the homotrimeric PCNA of humans but with three unique protein-binding sites. In the absence of nicked DNA, the
Sulfolobus solfataricus DNA ligase has an open, extended conformation. When complexed with heterotrimeric PCNA, the DNA ligase binds to the PCNA3 subunit and ligase retains an open, extended conformation. A closed, ring-shaped conformation of ligase catalyzes a DNA end-joining reaction that is strongly stimulated by PCNA. This open-to-closed switch in the conformation of DNA ligase is accommodated by a malleable interface with PCNA that serves as an efficient platform for DNA ligation.
We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation ...from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation.
DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle, while achieving error frequencies in the range of 10(-5) to 10(-6). Here we present a 2.2 A crystal ...structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer-template and a nucleoside triphosphate in the polymerase active site. The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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