Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.

Genetic screening identifies a SUMO protease dynamically maintaining centromeric chromatin.

Centromeres are defined by a self-propagating chromatin structure based on stable inheritance of CENP-A containing nucleosomes. Here, we present a genetic screen coupled to pulse-chase labeling that allow us to identify proteins selectively involved in deposition of nascent CENP-A or in long-term transmission of chromatin-bound CENP-A. These include factors with known roles in DNA replication, repair, chromatin modification, and transcription, revealing a broad set of chromatin regulators that impact on CENP-A dynamics. We further identify the SUMO-protease SENP6 as a key factor, not only controlling CENP-A stability but virtually the entire centromere and kinetochore. Loss of SENP6 results in hyper-SUMOylation of CENP-C and CENP-I but not CENP-A itself. SENP6 activity is required throughout the cell cycle, suggesting that a dynamic SUMO cycle underlies a continuous surveillance of the centromere complex that in turn ensures stable transmission of CENP-A chromatin.

A solid-phase transfection platform for arrayed CRISPR screens.

Arrayed CRISPR-based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid-phase transfection platform that enables CRISPR-based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high-throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies.

Catalytic mechanism of DNA-(cytosine-C5)-methyltransferases revisited: covalent intermediate formation is not essential for methyl group transfer by the murine Dnmt3a enzyme.

Co-transfections of reporter plasmids and plasmids encoding the catalytic domain of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene expression. As Dnmt3a mutants with C-->A and E-->A exchanges in the conserved PCQ and ENV motifs in the catalytic center of the enzyme also cause repression, we checked for their catalytic activity in vitro. Surprisingly, the activity of the cysteine variant and of the corresponding full-length Dnmt3a variant is only two to sixfold reduced with respect to wild-type Dnmt3a. In contrast, enzyme variants carrying E-->A, E-->D or E-->Q exchanges of the ENV glutamate are catalytically almost inactive, demonstrating that this residue has a central function in catalysis. Since the glutamic acid residue contacts the flipped base, its main function could be to hold the target base at a position that supports methyl group transfer. Whereas wild-type Dnmt3a and the ENV variants form covalent complexes with 5-fluorocytidine modified DNA, the PCN variant does not. Therefore, covalent complex formation is not essential in the reaction mechanism of Dnmt3a. We propose that correct positioning of the flipped base and the cofactor and binding to the transition state of methyl group transfer are the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl group transfer.

Specificity of DNA triple helix formation analyzed by a FRET assay.

BACKGROUND: A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis. RESULTS: We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 x 10(5) and 2.3 x 10(6) M(-1). Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg2+ ions whereas it is inhibited by K+ ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 x 10(4) and 8.1 x 10(4) min(-1) M(-1). Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation. CONCLUSION: We have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA.