Two distinct long-range synaptic complexes promote different aspects of end processing prior to repair of DNA breaks by non-homologous end joining.

Non-homologous end joining is the major double-strand break repair (DSBR) pathway in mammals. DNA-PK is the hub and organizer of multiple steps in non-homologous end joining (NHEJ). Recent high-resolution structures show how two distinct NHEJ complexes "synapse" two DNA ends. One complex includes a DNA-PK dimer mediated by XLF, whereas a distinct DNA-PK dimer forms via a domain-swap mechanism where the C terminus of Ku80 from one DNA-PK protomer interacts with another DNA-PK protomer in trans. Remarkably, the distance between the two synapsed DNA ends in both dimers is the same (∼115 Å), which matches the distance observed in the initial description of an NHEJ long-range synaptic complex. Here, a mutational strategy is used to demonstrate distinct cellular function(s) of the two dimers: one promoting fill-in end processing, while the other promotes DNA end resection. Thus, the specific DNA-PK dimer formed (which may be impacted by DNA end structure) dictates the mechanism by which ends will be made ligatable.

Unravelling the complexities of DNA-PK activation by structure-based mutagenesis.

It has been known for decades that the DNA-dependent protein kinase (DNA-PK) is only an active serine/threonine protein kinase when it is bound to a DNA double-stranded end; still, the molecular details of how this activation is achieved have remained elusive. The recent surge in structural information for DNA-PK complexes has provided valuable insights into the process of DNA end recognition by DNA-PK. A particularly intriguing feature of this kinase is a region of the protein that can transition from a seemingly structurally disordered state to a single alpha-helix that traverses down the DNA binding cradle. The DNA-PK bound DNA end of the DNA substrate engages with and appears to split around this helix which has been named the DNA End Blocking helix (DEB). Here a mutational approach is utilized to clarify the role of the DEB, and how DNA ends activate the enzyme. Our data suggest two distinct methods of kinase activation that is dependent on the DNA end chemistry. If the DNA end can split around the helix and stabilize the interaction between the DNA end and the DEB with a recently defined Helix-Hairpin-Helix (HHH) motif, the kinase forms an end-protection monomer that is active towards DNA-PK's many substrates. But if the DNA end cannot stably interact with the DEB [because of the DNA end structure, for instance hairpins, or because the DEB has been disrupted by mutation], the kinase is only partially activated, resulting in specific autophosphorylations of the DNA-PK monomer that allows nucleolytic end-processing. We posit that mutants that disrupt the capacity to stably generate the DEB/HHH DNA end-interaction are inefficient in generating the dimer complex that is requisite for NHEJ. In support of this idea, mutations that promote formation of this dimer partially rescue the severe cellular phenotypes associated with mutation of the DEB helix.

Catalytically inactive DNA ligase IV promotes DNA repair in living cells.

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that 'maximizes the fidelity of DNA repair'. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH - thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).

Autophosphorylation transforms DNA-PK from protecting to processing DNA ends.

The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.

Cryo-EM of NHEJ supercomplexes provides insights into DNA repair.

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.

Critical roles of Shugoshin and histones as tension sensors during mitosis.

Biorientation of paired sister chromosomes is required to maintain mitotic fidelity. A critical signal indicative of bipolar attachment is tension between cohesion-linked sister chromatids. Key components of the tension signaling apparatus include the Shugoshin family of proteins and the tension sensing motif of histone H3. Shugoshin proteins are recruited to chromatin to create discrete domains integral to tension sensing. Many factors involved in the chromatin association of Shugoshin proteins are well established, most strikingly through modifications found directly on centromeric and pericentric chromatin. It has been well established that phosphorylation at the centromere is essential to nucleating Shugoshin recruitment, but recent evidence revealed a role for pericentric histones and acetylation in modulating Shugoshin recruitment and activity. These data demonstrate that chromatins are not simply passive cargo during mitosis, but are instead actively involved in their segregation.

A Failsafe for Sensing Chromatid Tension in Mitosis with the Histone H3 Tail in Saccharomyces cerevisiae.

Mitotic fidelity is ensured by achieving biorientation on all paired chromosomes. The key signal for proper chromosome alignment is the tension between sister chromatids created by opposing poleward force from the spindles. In the budding yeast, the tension-sensing function requires that the Shugoshin protein, Shugoshin 1, be recruited to the centromeres and the neighboring pericentric regions. Concerted actions integrating proteins at centromeres and pericentromeres create highly specific Shugoshin 1 domains on mitotic chromosomes. We have previously reported that an important regulatory region on histone H3, termed the tension-sensing motif (TSM), is responsible for retaining Shugoshin 1 at pericentromeres. The TSM is negatively regulated by the acetyltransferase Gcn5p, but the underlying mechanism was elusive. In this work, we provide evidence that, when the TSM function is impaired, the histone H3 tail adopts a role that complements the damaged TSM to ensure faithful mitosis. This novel function of the H3 tail is controlled by Gcn5p, which targets selective lysine residues. Mutations to K14 and K23 ameliorate the mitotic defects resulting from TSM mutations. The restoration of faithful segregation is accompanied by regaining Shugoshin 1 access to the pericentric regions. Our data reveal a novel pathway for mitotic Shugoshin 1 recruitment and further reinforce the active role played by chromatins during their segregation in mitosis.

Correction: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

Resolving acetylated and phosphorylated proteins by neutral urea Triton-polyacrylamide gel electrophoresis: NUT-PAGE.

Protein acetylation and phosphorylation are key modifications that regulate both normal and pathological protein functions. The gel systems currently used for analyzing modified proteins require either expensive reagents or time-consuming second dimension electrophoresis. Here we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. The neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system separates proteins based on their charge at pH 7.0 and generates discrete bands from each acetylated and/or phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents and requires only a single dimension of electrophoresis. We demonstrate the effectiveness of this system by analyzing the phosphorylated species of an acidic protein, α-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative for resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge.

Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.