@article {49404, title = {Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation.}, journal = {Science (New York, N.Y.)}, volume = {361}, year = {2018}, month = {2018 Aug 24}, abstract = {

Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)-tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

}, issn = {1095-9203}, doi = {10.1126/science.aau1504}, author = {Vemu, Annapurna and Szczesna, Ewa and Zehr, Elena A and Spector, Jeffrey O and Grigorieff, Nikolaus and Deaconescu, Alexandra M and Roll-Mecak, Antonina} } @article {45184, title = {Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase.}, journal = {Cell}, volume = {157}, year = {2014}, month = {2014 Jun 5}, pages = {1405-15}, abstract = {

Acetylation of α-tubulin Lys40 by tubulin acetyltransferase (TAT) is the only known posttranslational modification in the microtubule lumen. It marks stable microtubules and is required for polarity establishment and directional migration. Here, we elucidate the mechanistic underpinnings for TAT activity and its preference for microtubules with slow turnover. 1.35\ {\r A} TAT cocrystal structures with bisubstrate analogs constrain TAT action to the microtubule lumen and reveal Lys40 engaged in a suboptimal active site. Assays with diverse tubulin polymers show that TAT is stimulated by microtubule interprotofilament contacts. Unexpectedly, despite the confined intraluminal location of Lys40, TAT efficiently scans the microtubule bidirectionally and acetylates stochastically without preference for ends. First-principles modeling and single-molecule measurements demonstrate that TAT catalytic activity, not constrained luminal diffusion, is rate limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2014.03.061}, author = {Szyk, Agnieszka and Deaconescu, Alexandra M and Spector, Jeffrey and Goodman, Benjamin and Valenstein, Max L and Ziolkowska, Natasza E and Kormendi, Vasilisa and Grigorieff, Nikolaus and Roll-Mecak, Antonina} } @article {6327, title = {Interplay of DNA repair with transcription: from structures to mechanisms.}, journal = {Trends in Biochemical Sciences}, volume = {37}, year = {2012}, month = {2012 Dec}, pages = {543-52}, abstract = {

Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions.

}, issn = {0968-0004}, doi = {10.1016/j.tibs.2012.09.002}, author = {Deaconescu, Alexandra M and Artsimovitch, Irina and Grigorieff, Nikolaus} } @article {6329, title = {Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 Feb 28}, pages = {3353-8}, abstract = {

Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.

}, keywords = {Adenosine Triphosphatases, Adenosine Triphosphate, Amino Acid Sequence, Bacterial, Bacterial Proteins, Crystallography, DNA, DNA Damage, DNA Helicases, DNA Repair, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Hydrolysis, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Conformation, Protein Interaction Mapping, Protein Structure, RNA Polymerase I, Tertiary, Transcription Factors, X-Ray}, issn = {1091-6490}, doi = {10.1073/pnas.1115105109}, author = {Deaconescu, Alexandra M and Sevostyanova, Anastasia and Artsimovitch, Irina and Grigorieff, Nikolaus} } @article {6337, title = {Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1.}, journal = {The Journal of Cell Biology}, volume = {189}, year = {2010}, month = {2010 Jun 28}, pages = {1087-96}, abstract = {

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal "basic" domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly-promoting factors with complementary activities.

}, keywords = {Actin Capping Proteins, Actins, Adenomatous Polyposis Coli Protein, Animals, Carrier Proteins, Fetal Proteins, Mice, Microfilament Proteins, NIH 3T3 Cells, Nuclear Proteins, Profilins, Protein Multimerization, Protein Transport}, issn = {1540-8140}, doi = {10.1083/jcb.201001016}, author = {Okada, Kyoko and Bartolini, Francesca and Deaconescu, Alexandra M and Moseley, James B and Dogic, Zvonimir and Grigorieff, Nikolaus and Gundersen, Gregg G and Goode, Bruce L} }