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Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability., Competing Interests: Declaration of interests K.A.C. is a member of the scientific advisory board of IDEAYA Biosciences and RADD Pharmaceuticals and is on the oncology advisory board for GlaxoSmithKline. S.J.B. is a co-founder, VP Science Strategy and shareholder at Artios Pharma Ltd. K.A.C. and S.J.B. are also members of the advisory board at Molecular Cell., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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, comprising an RNA-DNA hybrid and a displaced strand of ssDNA. R-loops have important physiological roles in cells, but deregulation of R-loop dynamics can also have harmful cellular outcomes. The genome-wide mapping of R-loops offers an unbiased approach to study R-loop biology in a wide range of contexts. Here we present a protocol to sequence RNA-DNA hybrids genome-wide with strand-specificity and high resolution. We also include information on how to prepare and incorporate into the workflow appropriate internal spike-in standards which facilitate accurate normalization of the sequencing signal, thereby providing quantitative insights into R-loop formation between different experimental samples., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G 1 /S, G 2 /M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G 2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)-directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G 2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G 2 checkpoint., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.)

One way to preserve a rare book is to lock it away from all potential sources of damage. Of course, an inaccessible book is also of little use, and the paper and ink will continue to degrade with age in any case. Like a book, the information stored in our DNA needs to be read, but it is also subject to continuous assault and therefore needs to be protected. In this Review, we examine how the replication stress response that is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR) senses and resolves threats to DNA integrity so that the DNA remains available to read in all of our cells. We discuss the multiple data that have revealed an elegant yet increasingly complex mechanism of ATR activation. This involves a core set of components that recruit ATR to stressed replication forks, stimulate kinase activity and amplify ATR signalling. We focus on the activities of ATR in the control of cell cycle checkpoints, origin firing and replication fork stability, and on how proper regulation of these processes is crucial to ensure faithful duplication of a challenging genome.

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.)

Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

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.

R-loops, nucleic acid structures consisting of an RNA-DNA hybrid and displaced single-stranded (ss) DNA, are ubiquitous in organisms from bacteria to mammals. First described in bacteria where they initiate DNA replication, it now appears that R-loops regulate diverse cellular processes such as gene expression, immunoglobulin (Ig) class switching, and DNA repair. Changes in R-loop regulation induce DNA damage and genome instability, and recently it was shown that R-loops are associated with neurodegenerative disorders. We discuss recent developments in the field; in particular, the regulation and effects of R-loops in cells, their effect on genomic and epigenomic stability, and their potential contribution to the origin of diseases including cancer and neurodegenerative disorders., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.)

Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

Replication stress is a complex phenomenon that has serious implications for genome stability, cell survival and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATR (ATM- and Rad3-related). Along with its downstream effectors, ATR stabilizes and helps to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding this response may be key to diagnosing and treating human diseases caused by defective responses to replication stress.

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.

Mutations can be beneficial under conditions in which genetic diversity is advantageous, such as somatic hypermutation and antibody generation, but they can also be lethal when they disrupt basic cellular processes or cause uncontrolled proliferation and cancer. Mutations arise from inaccurate processing of lesions generated by endogenous and exogenous DNA damaging agents, and the genome is particularly vulnerable to such damage during S phase. In this phase of the cell cycle, many lesions in the DNA template block replication. Such lesions must be bypassed in order to preserve fork stability and to ensure completion of DNA replication. Lesion bypass is carried out by a set of error-prone and error-free processes collectively referred to as DNA damage tolerance mechanisms. Here, we discuss how two types of DNA damage tolerance, translesion synthesis and template switching, are regulated at stalled replication forks by ubiquitination of PCNA, and the conditions under which they occur.

Genome maintenance is a constant concern for cells, and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases act as master regulators of the DNA-damage response by signalling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Recent studies have provided new insights into the mechanisms that control ATR activation, have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of ATR in maintaining genome integrity.

The ATR kinase is a critical upstream component of a checkpoint pathway that responds to many forms of damaged and incompletely replicated DNA. Cellular processes such as DNA replication and repair are thought to convert these DNA lesions into a common DNA intermediate that activates this signaling pathway. Indeed, numerous studies have shown that two DNA structures formed during these processes--single-stranded DNA (ssDNA) and junctions between double-stranded DNA (dsDNA) and ssDNA--are important components of the ATR-activating structure. However, an unanswered question is whether primed ssDNA is sufficient for activation of the ATR response. We recently demonstrated that primed ssDNA is sufficient to induce a bona fide checkpoint response in Xenopus egg extracts. This is the first well-defined DNA structure capable of eliciting ATR activation. Using this structure, we examined the contribution of ds/ssDNA junctions and ssDNA to checkpoint activation. Our results indicate the context in which the checkpoint-activating structure is generated may contribute significantly to its signaling properties. Here we discuss the implications of our findings, in the context of other recent work in the field, on our understanding of checkpoint signaling.