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In recent years, increasing evidence has highlighted the profound connection between DNA damage repair and the activation of immune responses. We spoke with researchers about their mechanistic interplays and the implications for cancer and other diseases., Competing Interests: Declaration of interests Karlene A. Cimprich is a member of the scientific advisory board of IDEAYA Biosciences, the scientific advisory board at RADD Pharmaceuticals, and the advisory board of Molecular Cell. Sandra Demaria has received compensation for consultant/advisory services from Lytix Biopharma, EMD Serono, Ono Pharmaceutical, Genentech, and Johnson & Johnson Enterprise Innovation Inc. and research support from Lytix Biopharma, Nanobiotix, and Boehringer-Ingelheim for unrelated projects., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Transcription-replication collisions (TRCs) are crucial determinants of genome instability. R-loops were linked to head-on TRCs and proposed to obstruct replication fork progression. The underlying mechanisms, however, remained elusive due to the lack of direct visualization and of non-ambiguous research tools. Here, we ascertained the stability of estrogen-induced R-loops on the human genome, visualized them directly by electron microscopy (EM), and measured R-loop frequency and size at the single-molecule level. Combining EM and immuno-labeling on locus-specific head-on TRCs in bacteria, we observed the frequent accumulation of DNA:RNA hybrids behind replication forks. These post-replicative structures are linked to fork slowing and reversal across conflict regions and are distinct from physiological DNA:RNA hybrids at Okazaki fragments. Comet assays on nascent DNA revealed a marked delay in nascent DNA maturation in multiple conditions previously linked to R-loop accumulation. Altogether, our findings suggest that TRC-associated replication interference entails transactions that follow initial R-loop bypass by the replication fork., (© 2023. The Author(s).)

R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability 1 , which has been linked to the action of endonucleases such as XPG 2-4 . However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1 (refs. 5-7 ), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref.  8 ), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (ref.  9 ) and in BRCA1-mutated cancer cells 10 . These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Although transcription is an essential cellular process, it is paradoxically also a well-recognized cause of genomic instability. R-loops, non-B DNA structures formed when nascent RNA hybridizes to DNA to displace the non-template strand as single-stranded DNA (ssDNA), are partially responsible for this instability. Yet, recent work has begun to elucidate regulatory roles for R-loops in maintaining the genome. In this review, we discuss the cellular contexts in which R-loops contribute to genomic instability, particularly during DNA replication and double-strand break (DSB) repair. We also summarize the evidence that R-loops participate as an intermediate during repair and may influence pathway choice to preserve genomic integrity. Finally, we discuss the immunogenic potential of R-loops and highlight their links to disease should they become pathogenic., Competing Interests: Declaration of interests Karlene A. Cimprich is a member of the Advisory Board for Molecular Cell., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replication–associated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1–Cul1–F-box protein (SCF) ubiquitin ligase complex and regulator of key cell-cycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin F–SCF complex, represses premature stem cell quiescence exit via ubiquitin–proteasome degradation of these factors.

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions., (© 2021 Crossley et al.)

Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.

R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes but can also cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA-DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here, we describe quantitative differential DNA-RNA immunoprecipitation (qDRIP), a method combining synthetic RNA-DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. We also use these quantitative comparisons to make the first estimates of the absolute count of RNA-DNA hybrids per cell and their half-lives genome-wide. Finally, we identify a subset of RNA-DNA hybrids with high GC skew which are partially resistant to RNase H. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

During transcription, the nascent RNA strand can base pair with its template DNA, displacing the non-template strand as ssDNA and forming a structure called an R-loop. R-loops are common across many domains of life and cause DNA damage in certain contexts. In this review, we summarize recent results implicating R-loops as important regulators of cellular processes such as transcription termination, gene regulation, and DNA repair. We also highlight recent work suggesting that R-loops can be problematic to cells as blocks to efficient transcription and replication that trigger the DNA damage response. Finally, we discuss how R-loops may contribute to cancer, neurodegeneration, and inflammatory diseases and compare the available next-generation sequencing-based approaches to map R-loops genome wide., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor γ (PPARγ) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARγ promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARγ depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARγ DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARγ signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARγ DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Faithful DNA replication is challenged by stalling of replication forks during S phase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed S phase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout S phase. Moreover, cells that re-enter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged S phase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout S phase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events., (Copyright © 2018. Published by Elsevier Inc.)

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The complex machineries involved in replication and transcription translocate along the same DNA template, often in opposing directions and at different rates. These processes routinely interfere with each other in prokaryotes, and mounting evidence now suggests that RNA polymerase complexes also encounter replication forks in higher eukaryotes. Indeed, cells rely on numerous mechanisms to avoid, tolerate, and resolve such transcription-replication conflicts, and the absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability. In this article, we review the cellular responses to transcription-replication conflicts and highlight how these inevitable encounters shape the genome and impact diverse cellular processes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility., Competing Interests: The authors declare that no competing interests exist.

Juvenile ciliopathy syndromes that are associated with renal cysts and premature renal failure are commonly the result of mutations in the gene encoding centrosomal protein CEP290. In addition to centrosomes and the transition zone at the base of the primary cilium, CEP290 also localizes to the nucleus; however, the nuclear function of CEP290 is unknown. Here, we demonstrate that reduction of cellular CEP290 in primary human and mouse kidney cells as well as in zebrafish embryos leads to enhanced DNA damage signaling and accumulation of DNA breaks ex vivo and in vivo. Compared with those from WT mice, primary kidney cells from Cep290-deficient mice exhibited supernumerary centrioles, decreased replication fork velocity, fork asymmetry, and increased levels of cyclin-dependent kinases (CDKs). Treatment of Cep290-deficient cells with CDK inhibitors rescued DNA damage and centriole number. Moreover, the loss of primary cilia that results from CEP290 dysfunction was rescued in 3D cell culture spheroids of primary murine kidney cells after exposure to CDK inhibitors. Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes.

Stalled replication forks are a critical problem for the cell because they can lead to complex genome rearrangements that underlie cell death and disease. Processes such as DNA damage tolerance and replication fork reversal protect stalled forks from these events. A central mediator of these DNA damage responses in humans is the Rad5-related DNA translocase, HLTF. Here, we present biochemical and structural evidence that the HIRAN domain, an ancient and conserved domain found in HLTF and other DNA processing proteins, is a modified oligonucleotide/oligosaccharide (OB) fold that binds to 3' ssDNA ends. We demonstrate that the HIRAN domain promotes HLTF-dependent fork reversal in vitro through its interaction with 3' ssDNA ends found at forks. Finally, we show that HLTF restrains replication fork progression in cells in a HIRAN-dependent manner. These findings establish a mechanism of HLTF-mediated fork reversal and provide insight into the requirement for distinct fork remodeling activities in the cell., (Copyright © 2015 Elsevier Inc. All rights reserved.)

R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability; however, the mechanisms underlying R-loop-induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Deoxyribonucleic acid (DNA) lesions encountered during replication are often bypassed using DNA damage tolerance (DDT) pathways to avoid prolonged fork stalling and allow for completion of DNA replication. Rad18 is a central E3 ubiquitin ligase in DDT, which exists in a monoubiquitinated (Rad18•Ub) and nonubiquitinated form in human cells. We find that Rad18 is deubiquitinated when cells are treated with methyl methanesulfonate or hydrogen peroxide. The ubiquitinated form of Rad18 does not interact with SNF2 histone linker plant homeodomain RING helicase (SHPRH) or helicase-like transcription factor, two downstream E3 ligases needed to carry out error-free bypass of DNA lesions. Instead, it interacts preferentially with the zinc finger domain of another, nonubiquitinated Rad18 and may inhibit Rad18 function in trans. Ubiquitination also prevents Rad18 from localizing to sites of DNA damage, inducing proliferating cell nuclear antigen monoubiquitination, and suppressing mutagenesis. These data reveal a new role for monoubiquitination in controlling Rad18 function and suggest that damage-specific deubiquitination promotes a switch from Rad18•Ub-Rad18 complexes to the Rad18-SHPRH complexes necessary for error-free lesion bypass in cells., (© 2014 Zeman et al.)

Rationale: Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disorder characterized by progressive loss of pulmonary microvessels. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable and ∼15% of patients with IPAH, their low penetrance (∼20%) suggests that other unidentified genetic modifiers are required for manifestation of the disease phenotype. Use of whole-exome sequencing (WES) has recently led to the discovery of novel susceptibility genes in heritable PAH, but whether WES can also accelerate gene discovery in IPAH remains unknown., Objectives: To determine whether WES can help identify novel gene modifiers in patients with IPAH., Methods: Exome capture and sequencing was performed on genomic DNA isolated from 12 unrelated patients with IPAH lacking BMPR2 mutations. Observed genetic variants were prioritized according to their pathogenic potential using ANNOVAR., Measurements and Main Results: A total of nine genes were identified as high-priority candidates. Our top hit was topoisomerase DNA binding II binding protein 1 (TopBP1), a gene involved in the response to DNA damage and replication stress. We found that TopBP1 expression was reduced in vascular lesions and pulmonary endothelial cells isolated from patients with IPAH. Although TopBP1 deficiency made endothelial cells susceptible to DNA damage and apoptosis in response to hydroxyurea, its restoration resulted in less DNA damage and improved cell survival., Conclusions: WES led to the discovery of TopBP1, a gene whose deficiency may increase susceptibility to small vessel loss in IPAH. We predict that use of WES will help identify gene modifiers that influence an individual's risk of developing IPAH.

Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.

The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia telangiectasia mutated). We reveal a function for MRN in ATR (ATM- and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required for recruitment of TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction and that this recruitment is necessary for phosphorylation of CHK1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at an ssDNA-to-dsDNA junction, 911 is required for TOPBP1 "activation." These findings provide molecular insights into how ATR is activated., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Studies from Ciccia et al. (2012) and Yuan et al. (2012) in this issue of Molecular Cell, together with Weston et al. (2012), reveal that the translocase ZRANB3/AH2 can recognize K63-linked polyubiquitinated PCNA and plays an important role in restarting stalled replication forks., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Postreplication 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 η, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase κ. Our data suggest not only 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Stalled replication forks activate and are stabilized by the ATR (ataxia-telangiectasia mutated and Rad3 related)-mediated checkpoint, but ultimately, they must also recover from the arrest. Although primed single-stranded DNA (ssDNA) is sufficient for checkpoint activation, it is still unknown how this signal is generated at a stalled replication fork. Furthermore, it is not clear how recovery and fork restart occur in higher eukaryotes. Using Xenopus laevis egg extracts, we show that DNA replication continues at a stalled fork through the synthesis and elongation of new primers independent of the checkpoint. This synthesis is dependent on the activity of proliferating cell nuclear antigen, Pol-delta, and Pol-epsilon, and it contributes to the phosphorylation of Chk1. We also used defined DNA structures to show that for a fixed amount of ssDNA, increasing the number of primer-template junctions strongly enhances Chk1 phosphorylation. These results suggest that new primers are synthesized at stalled replication forks by the leading and lagging strand polymerases and that accumulation of these primers may contribute to checkpoint activation.

In this issue of Genes & Development, four papers report that the annealing helicase HepA-related protein (HARP, also known as SMARCAL1 [SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1]) binds directly to the ssDNA-binding protein Replication protein A (RPA) and is recruited to sites of replicative stress. Knockdown of HARP results in hypersensitivity to multiple DNA-damaging agents and defects in fork stability or restart. These exciting insights reveal a key new player in the S-phase DNA damage response.

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 additional 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 downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed 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 gammaH2AX 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.

Proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around double-stranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates.

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.

Proliferating cell nuclear antigen (PCNA) is a homotrimeric, ring-shaped protein complex that functions as a processivity factor for DNA polymerases. Following genotoxic stress, PCNA is modified at a conserved site by either a single ubiquitin moiety or a polyubiquitin chain. These modifications are required to coordinate DNA damage tolerance processes with ongoing replication. The molecular mechanisms responsible for inducing PCNA ubiquitination are not well understood. Using Xenopus egg extracts, we show that ultraviolet radiation and aphidicolin treatment induce the mono- and diubiquitination of PCNA. PCNA ubiquitination is replication-dependent and coincides with activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage checkpoint pathway. However, loss of ATR signaling by depletion of the ATR-interacting protein (ATRIP) or Rad1, a component of the 911 checkpoint clamp, does not impair PCNA ubiquitination. Primed single-stranded DNA generated by uncoupling of mini-chromosome maintenance helicase and DNA polymerase activities has been shown previously to be necessary for ATR activation. Here we show that PCNA ubiquitination also requires uncoupling of helicase and polymerase activities. We further demonstrate that replicating single-stranded DNA, which mimics the structure produced upon uncoupling, is sufficient to induce PCNA monoubiquitination. Our results suggest that PCNA ubiquitination and ATR activation are two independent events that occur in response to a common single-stranded DNA intermediate generated by functional uncoupling of mini-chromosome maintenance (MCM) helicase and DNA polymerase activities.

An essential component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-activating structure is single-stranded DNA. It has been suggested that nucleotide excision repair (NER) can lead to activation of ATR by generating such a signal, and in yeast, DNA damage processing through the NER pathway is necessary for checkpoint activation during G1. We show here that ultraviolet (UV) radiation-induced ATR signaling is compromised in XPA-deficient human cells during S phase, as shown by defects in ATRIP (ATR-interacting protein) translocation to sites of UV damage, UV-induced phosphorylation of Chk1 and UV-induced replication protein A phosphorylation and chromatin binding. However, ATR signaling was not compromised in XPC-, CSB-, XPF- and XPG-deficient cells. These results indicate that damage processing is not necessary for ATR-mediated S-phase checkpoint activation and that the lesion recognition function of XPA may be sufficient. In contrast, XP-V cells deficient in the UV bypass polymerase eta exhibited enhanced ATR signaling. Taken together, these results suggest that lesion bypass and not lesion repair may raise the level of UV damage that can be tolerated before checkpoint activation, and that XPA plays a critical role in this activation.

The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.

Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

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 Polalpha 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.

Cells mount a coordinated response to DNA damage, activating DNA repair pathways and cell-cycle checkpoint pathways to allow time for DNA repair to occur. In human cells, checkpoint responses can be divided into p53-dependent and p53-independent pathways, the latter being predominant in G2 phase of the cell cycle. The p53-independent pathway involves a phosphorylation cascade that activates the Chk1 effector kinase and induces G2 arrest through inhibitory tyrosine phosphorylation of Cdc2. At the top of this cascade are the ATR and ATM kinases. How ATM and ATR recognize DNA damage and activate this checkpoint pathway is only beginning to emerge. Single-stranded DNA, a result of stalled DNA replication or processing of chromosomal lesions, appears to be central to the activation of ATR. The recruitment of replication protein A to single-stranded DNA facilitates the recruitment of several complexes of checkpoint proteins. In this context, ATR is activated and then phosphorylates the C-terminus of Chk1, activating it to enforce a block to mitotic entry.

Adaptation to the DNA damage checkpoint is a phenomenon long thought to be confined to the unicellular world. A new report in this issue of Cell by suggests the presence of a checkpoint adaptation pathway in Xenopus egg extracts that displays interesting molecular parallels to adaptation in yeast.

The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.

Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.

Common fragile sites are specific regions in the human genome that are particularly prone to genomic instability under conditions of replicative stress. Recent data suggest that these sites depend on the checkpoint kinase ATR to maintain their stability.

Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore the fundamental importance of proximity in enzyme regulation and of keeping a proper distance for maintaining signaling specificity.

We have analyzed how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase. The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase.