Poly(ADP-ribose) polymerase I (PARP1) is a primary DNA damage sensor whose (ADP-ribose) polymerase activity is acutely regulated by interaction with DNA breaks. Upon activation at sites of DNA damage, PARP1 modifies itself and other proteins by covalent addition of long branched polymers of ADP-ribose, which in turn recruit downstream DNA repair and chromatin remodelling factors. PARP1 recognizes DNA damage through its N-terminal DNA-binding domain (DBD), which consists of a tandem repeat of an unusual zinc-finger (ZnF) domain. We have now determined the crystal structure of the human PARP1-DBD bound to a DNA break. Along with functional analysis of PARP1 recruitment to sites of DNA damage in vivo, the structure reveals a dimeric assembly whereby ZnF1 and ZnF2 domains from separate PARP1 molecules form a strand-break recognition module that helps activate PARP1 by facilitating its dimerization and consequent trans-automodification.Short-patch repair of DNA single-strand breaks is initiated by poly(ADP-ribose) polymerase-1 (PARP1) -a multi-domain enzyme activated by binding of its N-terminal DNA-binding domain (DBD) to DNA breaks [1][2][3][4][5] . Activated PARP1 utilises NAD + to Correspondence to: Andreas G. Ladurner; Laurence H. Pearl; Antony W. Oliver. AUTHOR CONTRIBUTIONS A.A.E.A. purified the protein, crystallized the complex and collected the X-ray diffraction data; G.T. designed and constructed the PARP1-EGFP constructs and performed the laser DNA damage experiments; M.K. performed the FRAP experiments; P.O.H. engineered the knockdown PARP1 cell line and the wild-type imaging reporter constructs, and performed the in vitro complementation assays; M.H. and R.A.-B. engineered and purified mutant PARP1 constructs; A.G.L. designed the study and analysed the data; L.H.P designed the study, analysed the data and wrote the paper; A.W.O. made the baculovirus constructs, designed the purification protocol, and solved and refined the crystal structure. All authors discussed the results and commented on the manuscript. We have now determined the crystal structure of the DNA-binding domain of PARP1 (PARP1-DBD) encompassing the first two zinc-finger (ZnF) domains, bound to a DNA break. In contrast with structural analysis of the separated domains 22 , we show that DNA binding by both zinc-finger domains is essential to damage recruitment in vivo, and that ZnF1 and ZnF2 domains from separate PARP1 molecules act as a functional unit to generate a dimeric binding module that specifically recognizes the single-strand / double-strand transition at a recessed DNA break. Mutational analysis in vitro and in cells demonstrates the functional requirement for zinc-finger dimerisation and reveals a mechanism for bringing two PARP1 molecules into close proximity at a DNA break as a prerequisite for transmodification. RESULTS Structure of the PARP1-DBD -DNA ComplexAn N-terminal segment of human PARP1 (residues 5-202) was expressed in insect cells and purified by column chromatography. Screening with a range of DNA molecules...

Mutations in the MTM1 gene encoding myotubularin cause X-linked myotubular myopathy (XLMTM), a well-defined subtype of human centronuclear myopathy. Seven male Labrador Retrievers, age 14-26 wk, were clinically evaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure, and mitochondrial accumulations. Wild-type triads were infrequent, with most exhibiting an abnormal orientation of T tubules. MTM1 gene sequencing revealed a unique exon 7 variant in all seven affected males, causing a nonconservative missense change, p.N155K, which haplotype data suggest derives from a recent founder in the local population. Analysis of a worldwide panel of 237 unaffected Labrador Retrievers and 59 additional control dogs from 25 other breeds failed to identify this variant, supporting it as the pathogenic mutation. Myotubularin protein levels and localization were abnormal in muscles from affected dogs, and expression of GFP-MTM1 p.N155K in COS-1 cells showed that the mutant protein was sequestered in proteasomes, where it was presumably misfolded and prematurely degraded. These data demonstrate that XLMTM in Labrador Retrievers is a faithful genetic model of the human condition.congenital myopathy | myotubularin | necklace fibers | canine myopathy | animal model X -linked myotubular myopathy (XLMTM) is a well-defined subgroup of the centronuclear myopathies (CNMs) characterized by early onset and the presence of uniformly small muscle fibers with centrally placed nuclei resembling fetal myotubes (1, 2). Although centrally located nuclei can be found in many myopathies, clinical, genetic, and pathological factors can help distinguish these myopathies from XLMTM. Onset of clinical signs is typically at or near birth, and affected males have profound hypotonia and weakness accompanied by respiratory difficulties that usually require ventilatory support. The defective gene, MTM1, was identified in 1996 by positional cloning (3). Myotubularin, the protein encoded by the MTM1 gene, is a ubiquitously expressed phosphoinositide phosphatase implicated in intracellular vesicle trafficking and autophagy (4, 5). In skeletal muscle, myotubularin localizes to the triadic regions, where it likely plays a role in lipid biogenesis or metabolism (6).Animal models have played an important role in understanding the pathogenesis of how loss of MTM1 function leads to clinically evident myotubular myopathy. A classical knockout (KO) for the murine Mtm1 gene showed that myotubularin-deficient mice developed a progressive CNM during postnatal life that severely reduced life expectancy (7). Studies in this model, as well as in a related muscle-specific KO line, have demonstrated that myotubularin plays a role in muscle maintenance rather than maturation, and have c...

PARP1 and its effector, the ATP-dependent chromatin remodeler Alc1/Chd1L, are identified as key players during the rapid chromatin relaxation at DNA damage sites.

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP-ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1-dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform-specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.

Oncogene activation is usually not enough to induce cancer, but causes cells to arrest proliferation, alter chromatin structure, and increase protein secretion. In this issue of Molecular Cell, Chen et al. (2015) implicate the histone variant macroH2A.1 in the regulation of senescence.

22In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense 23 systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have 24 previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells 25 from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), α-26 ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in 27 DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA 28 repair (TCR) on MMS-induced mutagenesis in E.coli strain deficient in functional AlkB protein.

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In the version of this article initially published, the image in the bottom row of Figure 4a (full-length PARP1-EGFP mutant R138E) was mistakenly replaced with a duplicate of the bottom-row image in Figure 4c (full-length PARP1-EGFP mutant M43D F44D) during preparation of the accepted version of the manuscript, after peer review and editorial evaluation had taken place. The error has been corrected in the HTML and PDF versions of the article. co r r i g e n Da npg