Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.

The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

Linkage reprogramming by tailor-made E3s reveals polyubiquitin chain requirements in DNA-damage bypass.

A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often poorly understood because of our inability to control or manipulate this parameter in vivo. Here, we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases. Using the polyubiquitylation of the budding yeast replication factor PCNA in response to DNA damage as a model case, we show that altering the features of a polyubiquitin chain in vivo can change the fate of the modified substrate. We also provide evidence for redundancy between distinct but structurally similar linkages, and we demonstrate by proof-of-principle experiments that the method can be generalized to targets beyond PCNA. Our study illustrates a promising approach toward the in vivo analysis of polyubiquitin signaling.

Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks.

Tracing DNA repair factors by fluorescence microscopy provides valuable information about how DNA damage processing is orchestrated within cells. Most repair pathways involve single-stranded DNA (ssDNA), making replication protein A (RPA) a hallmark of DNA damage and replication stress. RPA foci emerging during S phase in response to tolerable loads of polymerase-blocking lesions are generally thought to indicate stalled replication intermediates. We now report that in budding yeast they predominantly form far away from sites of ongoing replication, and they do not overlap with any of the repair centers associated with collapsed replication forks or double-strand breaks. Instead, they represent sites of postreplicative DNA damage bypass involving translesion synthesis and homologous recombination. We propose that most RPA and recombination foci induced by polymerase-blocking lesions in the replication template are clusters of repair tracts arising from replication centers by polymerase re-priming and subsequent expansion of daughter-strand gaps over the course of S phase.

Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.

DNA single-strand breaks (SSBs) are among the most common lesions in the genome, arising spontaneously and as intermediates of many DNA transactions. Nevertheless, in contrast to double-strand breaks (DSBs), their distribution in the genome has hardly been addressed in a meaningful way. We now present a technique based on genome-wide ligation of 3'-OH ends followed by sequencing (GLOE-Seq) and an associated computational pipeline designed for capturing SSBs but versatile enough to be applied to any lesion convertible into a free 3'-OH terminus. We demonstrate its applicability to mapping of Okazaki fragments without prior size selection and provide insight into the relative contributions of DNA ligase 1 and ligase 3 to Okazaki fragment maturation in human cells. In addition, our analysis reveals biases and asymmetries in the distribution of spontaneous SSBs in yeast and human chromatin, distinct from the patterns of DSBs.

PETISCO is a novel protein complex required for 21U RNA biogenesis and embryonic viability.

Piwi proteins are important for germ cell development in most animals. These proteins are guided to specific targets by small guide RNAs, referred to as piRNAs or 21U RNAs in Caenorhabditis elegans In this organism, even though genetic screens have uncovered 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1 (piRNA-induced silencing-defective 1), we here define a novel protein complex, PETISCO (PID-3, ERH-2, TOFU-6, and IFE-3 small RNA complex), that is required for 21U RNA biogenesis. PETISCO contains both potential 5' cap and 5' phosphate RNA-binding domains and interacts with capped 21U precursor RNA. We resolved the architecture of PETISCO and revealed a second function for PETISCO in embryonic development. This essential function of PETISCO is mediated not by PID-1 but by the novel protein TOST-1 (twenty-one U pathway antagonist). In contrast, TOST-1 is not essential for 21U RNA biogenesis. Both PID-1 and TOST-1 interact directly with ERH-2 using a conserved sequence motif. Finally, our data suggest a role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes a key complex for 21U RNA processing in C. elegans and strengthens the view that 21U RNA biogenesis is built on an snRNA-related pathway.

Ubiquitylation of the 9-1-1 checkpoint clamp is independent of rad6-rad18 and DNA damage.

A recent report proposed a function of the ubiquitin conjugation factors Rad6 and Rad18 comparable to the bacterial SOS response, controlling damage-induced transcriptional activation and contributing to checkpoint signaling. The relevant ubiquitylation target was identified as budding yeast Rad17, a subunit of the PCNA-like 9-1-1 checkpoint clamp. We report here that in fact all three subunits of the 9-1-1 complex are ubiquitylated. However, in contrast to previous results, we found modification of Rad17 to be independent of DNA damage, the Rad6-Rad18 complex, the putative acceptor site (lysine 197), and loading of the complex onto DNA. Consistently, we were unable to observe enhanced damage sensitivity or defects in checkpoint signaling in a rad17(K197R) mutant. Instead, our findings suggest that ubiquitylation of the 9-1-1 complex may be a background reaction that in some cases can mediate proteasomal degradation.

Fast friends - Ubiquitin-like modifiers as engineered fusion partners.

Ubiquitin and its relatives are major players in many biological pathways, and a variety of experimental tools based on biological chemistry or protein engineering is available for their manipulation. One popular approach is the use of linear fusions between the modifier and a protein of interest. Such artificial constructs can facilitate the understanding of the role of ubiquitin in biological processes and can be exploited to control protein stability, interactions and degradation. Here we summarize the basic design considerations and discuss the advantages as well as limitations associated with their use. Finally, we will refer to several published case studies highlighting the principles of how they provide insight into pathways ranging from membrane protein trafficking to the control of epigenetic modifications.

Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance.

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.

Spatial separation between replisome- and template-induced replication stress signaling.

Polymerase-blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single-stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re-priming downstream of lesions can give rise to daughter-strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9-dependent mechanism of damage signaling is distinct from the Mrc1-dependent, fork-associated response to replication stress induced by conditions such as nucleotide depletion or replisome-inherent problems, but reminiscent of replication-independent checkpoint activation by single-stranded DNA Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase-blocking lesions mainly emanates from Exo1-processed, postreplicative daughter-strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome- versus template-induced checkpoint signaling.

GTSF-1 is required for formation of a functional RNA-dependent RNA Polymerase complex in Caenorhabditis elegans.

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf-1 and characterized it in the context of the sRNA pathways of C. elegans We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying rrf-3 mutants. We show, both in vivo and in vitro, that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities.

Ubc13-Mms2 cooperates with a family of RING E3 proteins in budding yeast membrane protein sorting.

Polyubiquitin chains linked via lysine (K) 63 play an important role in endocytosis and membrane trafficking. Their primary source is the ubiquitin protein ligase (E3) Rsp5/NEDD4, which acts as a key regulator of membrane protein sorting. The heterodimeric ubiquitin-conjugating enzyme (E2), Ubc13-Mms2, catalyses K63-specific polyubiquitylation in genome maintenance and inflammatory signalling. In budding yeast, the only E3 proteins known to cooperate with Ubc13-Mms2 so far is a nuclear RING finger protein, Rad5, involved in the replication of damaged DNA. Here, we report a contribution of Ubc13-Mms2 to the sorting of membrane proteins to the yeast vacuole via the multivesicular body (MVB) pathway. In this context, Ubc13-Mms2 cooperates with Pib1, a FYVE-RING finger protein associated with internal membranes. Moreover, we identified a family of membrane-associated FYVE-(type)-RING finger proteins as cognate E3 proteins for Ubc13-Mms2 in several species, and genetic analysis indicates that the contribution of Ubc13-Mms2 to membrane trafficking in budding yeast goes beyond its cooperation with Pib1. Thus, our results widely implicate Ubc13-Mms2 as an Rsp5-independent source of K63-linked polyubiquitin chains in the regulation of membrane protein sorting.This article has an associated First Person interview with the first author of the paper.

Ubiquitin signalling in DNA replication and repair.

Post-translational modification by ubiquitin is best known for its role in targeting its substrates for regulated degradation. However, non-proteolytic functions of the ubiquitin system, often involving either monoubiquitylation or polyubiquitylation through Lys63-linked chains, have emerged in various cell signalling pathways. These two forms of the ubiquitin signal contribute to three different pathways related to the maintenance of genome integrity that are responsible for the processing of DNA double-strand breaks, the repair of interstrand cross links and the bypass of lesions during DNA replication.

Effects of chain length and geometry on the activation of DNA damage bypass by polyubiquitylated PCNA.

Ubiquitylation of the eukaryotic sliding clamp, PCNA, activates a pathway of DNA damage bypass that facilitates the replication of damaged DNA. In its monoubiquitylated form, PCNA recruits a set of damage-tolerant DNA polymerases for translesion synthesis. Alternatively, modification by K63-linked polyubiquitylation triggers a recombinogenic process involving template switching. Despite the identification of proteins interacting preferentially with polyubiquitylated PCNA, the molecular function of the chain and the relevance of its K63-linkage are poorly understood. Using genetically engineered mimics of polyubiquitylated PCNA, we have now examined the properties of the ubiquitin chain required for damage bypass in budding yeast. By varying key parameters such as the geometry of the junction, cleavability and capacity for branching, we demonstrate that either the structure of the ubiquitin-ubiquitin junction or its dynamic assembly or disassembly at the site of action exert a critical impact on damage bypass, even though known effectors of polyubiquitylated PCNA are not strictly linkage-selective. Moreover, we found that a single K63-junction supports substantial template switching activity, irrespective of its attachment site on PCNA. Our findings provide insight into the interrelationship between the two branches of damage bypass and suggest the existence of a yet unidentified, highly linkage-selective receptor of polyubiquitylated PCNA.

Ubiquitin, SUMO, and phosphate: how a trio of posttranslational modifiers governs protein fate.

In this issue of Molecular Cell, Guo et al. (2012) demonstrate how a series of sequential posttranslational modifications, phosphorylation, sumoylation, and ubiquitylation, cooperate to target human flap endonuclease FEN1 to degradation by the proteasome at the end of S phase.

The helicase Pif1 functions in the template switching pathway of DNA damage bypass.

Replication of damaged DNA is challenging because lesions in the replication template frequently interfere with an orderly progression of the replisome. In this situation, complete duplication of the genome is ensured by the action of DNA damage bypass pathways effecting either translesion synthesis by specialized, damage-tolerant DNA polymerases or a recombination-like mechanism called template switching (TS). Here we report that budding yeast Pif1, a helicase known to be involved in the resolution of complex DNA structures as well as the maturation of Okazaki fragments during replication, contributes to DNA damage bypass. We show that Pif1 expands regions of single-stranded DNA, so-called daughter-strand gaps, left behind the replication fork as a consequence of replisome re-priming. This function requires interaction with the replication clamp, proliferating cell nuclear antigen, facilitating its recruitment to damage sites, and complements the activity of an exonuclease, Exo1, in the processing of post-replicative daughter-strand gaps in preparation for TS. Our results thus reveal a novel function of a conserved DNA helicase that is known as a key player in genome maintenance.

Monoubiquitylation of histone H2B contributes to the bypass of DNA damage during and after DNA replication.

DNA lesion bypass is mediated by DNA damage tolerance (DDT) pathways and homologous recombination (HR). The DDT pathways, which involve translesion synthesis and template switching (TS), are activated by the ubiquitylation (ub) of PCNA through components of the RAD6-RAD18 pathway, whereas the HR pathway is independent of RAD18 However, it is unclear how these processes are coordinated within the context of chromatin. Here we show that Bre1, an ubiquitin ligase specific for histone H2B, is recruited to chromatin in a manner coupled to replication of damaged DNA. In the absence of Bre1 or H2Bub, cells exhibit accumulation of unrepaired DNA lesions. Consequently, the damaged forks become unstable and resistant to repair. We provide physical, genetic, and cytological evidence that H2Bub contributes toward both Rad18-dependent TS and replication fork repair by HR. Using an inducible system of DNA damage bypass, we further show that H2Bub is required for the regulation of DDT after genome duplication. We propose that Bre1-H2Bub facilitates fork recovery and gap-filling repair by controlling chromatin dynamics in response to replicative DNA damage.

Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance.

Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5's helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.

Functions of SUMO in the Maintenance of Genome Stability.

Like in most other areas of cellular metabolism, the functions of the ubiquitin-like modifier SUMO in the maintenance of genome stability are manifold and varied. Perturbations of global sumoylation causes a wide spectrum of phenotypes associated with defects in DNA maintenance, such as hypersensitivity to DNA-damaging agents, gross chromosomal rearrangements and loss of entire chromosomes. Consistent with these observations, many key factors involved in various DNA repair pathways have been identified as SUMO substrates. However, establishing a functional connection between a given SUMO target, the cognate SUMO ligase and a relevant phenotype has remained a challenge, mainly because of the difficulties involved in identifying important modification sites and downstream effectors that specifically recognize the target in its sumoylated state. This review will give an overview over the major pathways of DNA repair and genome maintenance influenced by the SUMO system and discuss selected examples of SUMO's actions in these pathways where the biological consequences of the modification have been elucidated.