The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Pol␣ is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.

An unexpectedly large fraction of genes in metazoans (human, mouse, zebrafish, worm, fruit fly) express high levels of circularized RNAs containing canonical exons. Here we report that circular RNA isoforms are found in diverse species whose most recent common ancestor existed more than one billion years ago: fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae), a plant (Arabidopsis thaliana), and protists (Plasmodium falciparum and Dictyostelium discoideum). For all species studied to date, including those in this report, only a small fraction of the theoretically possible circular RNA isoforms from a given gene are actually observed. Unlike metazoans, Arabidopsis, D. discoideum, P. falciparum, S. cerevisiae, and S. pombe have very short introns (∼100 nucleotides or shorter), yet they still produce circular RNAs. A minority of genes in S. pombe and P. falciparum have documented examples of canonical alternative splicing, making it unlikely that all circular RNAs are by-products of alternative splicing or ‘piggyback’ on signals used in alternative RNA processing. In S. pombe, the relative abundance of circular to linear transcript isoforms changed in a gene-specific pattern during nitrogen starvation. Circular RNA may be an ancient, conserved feature of eukaryotic gene expression programs.

SUMMARY Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify novel genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose down-regulation led to elevated levels of H2AX phosphorylation (γH2AX) and revealed new links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased γH2AX levels to the neurological disorder, Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

Summary Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature.

Here, we demonstrate that primed, single-stranded DNA (ssDNA) is sufficient for activation of the ATR-dependent checkpoint pathway in Xenopus egg extracts. Using this structure, we define the contribution of the 5-and 3-primer ends to Chk1 activation when replication is blocked and ongoing. In addition, we show that although ssDNA is not sufficient for checkpoint activation, the amount of ssDNA adjacent to the primer influences the level of Chk1 phosphorylation. These observations define the minimal DNA requirements for checkpoint activation and suggest that primed ssDNA represents a common checkpoint activating-structure formed following many types of damage. The cellular response to DNA damage and replication stress is essential for the maintenance of genomic stability (Zhou and Elledge 2000;Melo and Toczyski 2002). The ATR (ATM and Rad3-related) kinase plays a central role in this pathway and responds to many types of genotoxic stress. Many of the effects of ATR are mediated by the downstream effector kinase Chk1, which is phosphorylated and activated by the ATR-ATRIP (ATR-interacting protein) complex (Zhou and Elledge 2000;Melo and Toczyski 2002). Activation of Chk1 also requires the function of several other proteins. Among these are the Rad9-Hus1-Rad1 (9-1-1) complex, a PCNA-related complex that is recruited to damaged chromatin, enhances ATR activation, and binds primer-template junctions in vitro (Ellison and Stillman 2003;Parrilla-Castellar et al. 2004;Majka et al. 2006b). Also important are TopBP1 and Claspin. TopBP1 activates the kinase activity of the ATR-ATRIP complex, and Claspin may both activate Chk1 and recruit it to ATR (Kumagai and Dunphy 2000; Lee et al. 2005;Kumagai et al. 2006).Despite the growing knowledge of proteins involved in checkpoint processes, the precise structure responsible for checkpoint activation following DNA damage or replication inhibition is not known. Several studies suggest the lesions induced by ultraviolet radiation, methyl methanesulfonate, and cisplatin activate the checkpoint most efficiently in S phase (Lupardus et al. 2002;Stokes et al. 2002;Tercero et al. 2003;Ward et al. 2004;Marini et al. 2006). In these cases and upon treatment with the polymerase inhibitor aphidicolin it is thought that replication forks stall, generating a common checkpointactivating intermediate through uncoupling of helicase and polymerase activities (Walter and Newport 2000;Pacek and Walter 2004;Byun et al. 2005;Cortez 2005). This process leads to accumulation of replication protein A (RPA)-coated single-stranded DNA (ssDNA), a structure sufficient to recruit the ATR-ATRIP complex and essential for checkpoint activation . However, several studies indicate that ssDNA is not sufficient and that additional replication is required for checkpoint activation and for loading of the 9-1-1 complex (Michael et al. 2000;Stokes et al. 2002;You et al. 2002;Byun et al. 2005). These observations suggest the checkpoint-activating structure is comprised of at least two parts, RPA-coated ssDNA and a prime...

SUMMARY Post-replication repair (PRR) pathways play important roles in restarting stalled replication forks and regulating mutagenesis. In yeast, Rad5-mediated damage avoidance and Rad18-mediated translesion synthesis (TLS) are two forms of PRR. Two Rad5-related proteins, SHPRH and HLTF, have been identified in mammalian cells, but their specific roles in PRR are unclear. Here, we show that HLTF and SHPRH suppress mutagenesis in a damage-specific manner, preventing mutations induced by UV and MMS, respectively. Following UV, HLTF enhances PCNA monoubiquitination and recruitment of TLS polymerase eta, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase kappa. Our data not only suggest that cells differentially utilize HLTF and SHPRH for different forms of DNA damage, but also, surprisingly, that HLTF and SHPRH may coordinate the two main branches of PRR to choose the proper bypass mechanism for minimizing mutagenesis.

ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.

Root systems develop different root types that individually sense cues from their local environment and integrate this information with systemic signals. This complex multi-dimensional amalgam of inputs enables continuous adjustment of root growth rates, direction, and metabolic activity that define a dynamic physical network. Current methods for analyzing root biology balance physiological relevance with imaging capability. To bridge this divide, we developed an integrated-imaging system called Growth and Luminescence Observatory for Roots (GLO-Roots) that uses luminescence-based reporters to enable studies of root architecture and gene expression patterns in soil-grown, light-shielded roots. We have developed image analysis algorithms that allow the spatial integration of soil properties, gene expression, and root system architecture traits. We propose GLO-Roots as a system that has great utility in presenting environmental stimuli to roots in ways that evoke natural adaptive responses and in providing tools for studying the multi-dimensional nature of such processes.DOI: http://dx.doi.org/10.7554/eLife.07597.001