Durocher

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third G is compensated by newly introduced non-base-specific interactions, enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

Publication date: 23 April 2015
Source:Cell, Volume 161, Issue 3
Author(s): Gheorghe Chistol , Johannes C. Walter
The first event in the initiation of eukaryotic DNA replication is the recruitment of the MCM2-7 ATPase, the core of the replicative DNA helicase, to origins. Ticau et al. use single-molecule imaging to reveal how ORC, Cdc6, and Cdt1 cooperate to load MCM2-7 onto DNA, enabling bidirectional replication.

Teaser

The first event in the initiation of eukaryotic DNA replication is the recruitment of the MCM2-7 ATPase, the core of the replicative DNA helicase, to origins. Ticau et al. use single-molecule imaging to reveal how ORC, Cdc6, and Cdt1 cooperate to load MCM2-7 onto DNA, enabling bidirection replication.

The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders.

Publication date: 18 September 2014
Source:Molecular Cell, Volume 55, Issue 6
Author(s): Hind Ghezraoui , Marion Piganeau , Benjamin Renouf , Jean-Baptiste Renaud , Annahita Sallmyr , Brian Ruis , Sehyun Oh , Alan E. Tomkinson , Eric A. Hendrickson , Carine Giovannangeli , Maria Jasin , Erika Brunet
Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

Graphical abstract

Teaser

Ghezraoui et al. use designer nucleases (ZFNs, TALENs, CRISPR/Cas9, and nCas9) to introduce concurrent double-strand breaks on two chromosomes to study chromosomal translocation joining mechanisms in human cells. In contrast with previous studies in mice, canonical nonhomologous end-joining involving DNA ligase IV generates translocations, including a translocation found in cancers.

Publication date: 19 June 2014
Source:Cell, Volume 157, Issue 7
Author(s): Sergi Regot , Jacob J. Hughey , Bryce T. Bajar , Silvia Carrasco , Markus W. Covert
Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.

Graphical abstract

Teaser

Regot et al. generate biosensors for kinase activity in individual cells that convert phosphorylation to localization changes, allowing the simultaneous measurement of JNK, p38, and ERK activities.

The small molecule Remodelin can correct nuclear architecture defects and improve cellular fitness of progeric human cells. Authors: Delphine Larrieu, Sébastien Britton, Mukerrem Demir, Raphaël Rodriguez, Stephen P. Jackson

Astrophysics: Hydrogen river could fuel stars

Nature 506, 7486 (2014). doi:10.1038/506008c

The discovery of a faint filament of hydrogen gas streaming across space could help to explain how some galaxies maintain their pace of star formation.D. J. Pisano from West Virginia University in Morgantown used the Robert C. Byrd Green Bank Telescope to identify a

Nature Methods 10, 620 (2013). doi:10.1038/nmeth.2526

Authors: Kristofor Webb & Eric J Bennett

Two approaches to serially enrich protein post-translational modifications allow the detection of multiple modifications in a single biological sample using mass spectrometry.

Nature Structural & Molecular Biology. doi:10.1038/nsmb.2592

Authors: Odil Porrua & Domenico Libri

Nature Methods. doi:10.1038/nmeth.2519

Authors: Danielle L Swaney, Pedro Beltrao, Lea Starita, Ailan Guo, John Rush, Stanley Fields, Nevan J Krogan & Judit Villén

Nature Methods. doi:10.1038/nmeth.2518

Authors: Philipp Mertins, Jana W Qiao, Jinal Patel, Namrata D Udeshi, Karl R Clauser, D R Mani, Michael W Burgess, Michael A Gillette, Jacob D Jaffe & Steven A Carr

We report a mass spectrometry–based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells.

Zhao Zhang, Xiangdong Lv, Wen-chi Yin, Xiaoyun Zhang, Jing Feng, Wenqing Wu, Chi-chung Hui, Lei Zhang, Yun Zhao. The Cubitus interruptus (Ci)/Gli family of transcription factors can be degraded either completely or partially from a full-length form (Ci155/Gli^FL) to a truncated repressor (Ci75/Gli

Feng Wang, Damian C. Ekiert, Insha Ahmad, Wenli Yu, Yong Zhang, Omar Bazirgan, Ali Torkamani, Terje Raudsepp, Waithaka Mwangi, Michael F. Criscitiello, Ian A. Wilson, Peter G. Schultz, Vaughn V. Smider. Some species mount a robust antibody response despite having limited genome-encoded combinatorial diversity potential. Cows are unusual in having exceptionally long CDR H3 loops and few V regions,....

Kirstin Keusekotten, Paul Ronald Elliott, Laura Glockner, Berthe Katrine Fiil, Rune Busk Damgaard, Yogesh Kulathu, Tobias Wauer, Manuela Kathrin Hospenthal, Mads Gyrd-Hansen, Daniel Krappmann, Kay Hofmann, David Komander. The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that specifically assembles Met1-linked (also known as linear) Ub chains that regulate nuclear factor κB (NF-κB) signaling.....

Tianlai Shi, Richard D. Bunker, Stefano Mattarocci, Cyril Ribeyre, Mahamadou Faty, Heinz Gut, Andrea Scrima, Ulrich Rass, Seth M. Rubin, David Shore, Nicolas H. Thomä. Yeast telomeres comprise irregular TG_1-3 DNA repeats bound by the general transcription factor Rap1. Rif1 and Rif2, along with Rap1, form the telosome, a protective cap that inhibits te....

Carlos López-Otín, Maria A. Blasco, Linda Partridge, Manuel Serrano, Guido Kroemer. Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death. This deterioration is the primary risk factor for major ....

Toyoaki Natsume, Carolin A. Müller, Yuki Katou, Renata Retkute, Marek Gierliński, Hiroyuki Araki, J. Julian Blow, Katsuhiko Shirahige, Conrad A. Nieduszynski, Tomoyuki U. Tanaka. Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister ch....

Nature Structural & Molecular Biology 20, 748 (2013). doi:10.1038/nsmb.2573

Authors: Richard T Pomerantz, Isabel Kurth, Myron F Goodman & Mike E O'Donnell

Nature Structural & Molecular Biology 20, 648 (2013). doi:10.1038/nsmb.2602

Authors: Elaine M Dunleavy, Weiguo Zhang & Gary H Karpen

Whether centromere-specific CENP-A–containing nucleosomes comprise one molecule each of CENP-A and histones H4, H2A and H2B (forming a tetramer or hemisome) or two molecules of all four histones (forming an octamer) has been controversial. Three new studies now address this controversy using complementary in vitro and in vivo approaches.

Nature advance online publication 05 June 2013. doi:10.1038/nature12274

Authors: Arik Cooper, Mayra García, Constantinos Petrovas, Takuya Yamamoto, Richard A. Koup & Gary J. Nabel

Human immunodeficiency virus-1 (HIV-1) has infected more than 60 million people and caused nearly 30 million deaths worldwide, ultimately the consequence of cytolytic infection of CD4+ T cells. In humans and in macaque models, most of these cells contain viral DNA and are rapidly eliminated at the peak of viraemia, yet the mechanism by which HIV-1 induces helper T-cell death has not been defined. Here we show that virus-induced cell killing is triggered by viral integration. Infection by wild-type HIV-1, but not an integrase-deficient mutant, induced the death of activated primary CD4 lymphocytes. Similarly, raltegravir, a pharmacologic integrase inhibitor, abolished HIV-1-induced cell killing both in cell culture and in CD4+ T cells from acutely infected subjects. The mechanism of killing during viral integration involved the activation of DNA-dependent protein kinase (DNA-PK), a central integrator of the DNA damage response, which caused phosphorylation of p53 and histone H2AX. Pharmacological inhibition of DNA-PK abolished cell death during HIV-1 infection in vitro, suggesting that processes which reduce DNA-PK activation in CD4 cells could facilitate the formation of latently infected cells that give rise to reservoirs in vivo. We propose that activation of DNA-PK during viral integration has a central role in CD4+ T-cell depletion, raising the possibility that integrase inhibitors and interventions directed towards DNA-PK may improve T-cell survival and immune function in infected individuals.

The structural links between the chromosomal centromere protein CenH3 and the kinetochore protein CENP-C are determined.

Authors: Hidenori Kato, Jiansheng Jiang, Bing-Rui Zhou, Marieke Rozendaal, Hanqiao Feng, Rodolfo Ghirlando, T. Sam Xiao, Aaron F. Straight, Yawen Bai

Nature Structural & Molecular Biology. doi:10.1038/nsmb.2585

Authors: Nicholas L Adkins, Hengyao Niu, Patrick Sung & Craig L Peterson

In yeast, the localization of homologous recombination–associated proteins to heterochromatic regions of the genome is necessary for proper nuclear organization.

J. Chad Brenner, Bushra Ateeq, Yong Li, Anastasia K. Yocum, Qi Cao, Irfan A. Asangani, Sonam Patel, Xiaoju Wang, Hallie Liang, Jindan Yu, Nallasivam Palanisamy, Javed Siddiqui, Wei Yan, Xuhong Cao, Rohit Mehra, Aaron Sabolch, Venkatesha Basrur, Robert J. Lonigro, Jun Yang, Scott A. Tomlins, Christopher A. Maher, Kojo S.J. Elenitoba-Johnson, Maha Hussain, Nora M. Navone, Kenneth J. Pienta, Sooryanarayana Varambally, Felix Y. Feng, Arul M. Chinnaiyan.

A modification on histone H3 in a nucleosome favors the swapping out of one type of H2A histone for another.

Authors: Shinya Watanabe, Marta Radman-Livaja, Oliver J. Rando, Craig L. Peterson