Transcription-Replication Conflicts as a Source of Genome Instability.

Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA sequence. Conflicts between the replication and transcription machineries (transcription-replication conflicts, or TRCs) are widespread in both prokaryotes and eukaryotes and have the capacity to both cause DNA damage and compromise complete, faithful replication of the genome. This review will highlight recent studies investigating the genomic locations of TRCs and the mechanisms by which they may be prevented, mitigated, or resolved. We address work from both model organisms and mammalian systems but predominantly focus on multicellular eukaryotes owing to the additional complexities inherent in the coordination of replication and transcription in the context of cell type-specific gene expression and higher-order chromatin organization.

A basal-level activity of ATR links replication fork surveillance and stress response.

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.

Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε.

Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.

TOP2ß-Dependent Nuclear DNA Damage Shapes Extracellular Growth Factor Responses via Dynamic AKT Phosphorylation to Control Virus Latency.

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.

In a Class of Its Own: A New Family of Deubiquitinases Promotes Genome Stability.

Several proteins are ubiquitylated in response to genotoxic stress; however, the roles of deubiquitinases (DUBs) in reversing these modifications are less well characterized. Two independent studies by Kwasna et al. (2018) and Haahr et al. (2018) identify a new type of cysteine protease DUB called ZUFSP, which cleaves K63-linked polyubiquitin chains at DNA damage sites to promote genome stability.

Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.

Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease.

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections.

ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress.

Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability, and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances, and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and link timely genome replication to DNA repair.

Transcription-replication conflicts as a source of common fragile site instability caused by BMI1-RNF2 deficiency.

Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcription-replication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity.

Fused in sarcoma regulates DNA replication timing and kinetics.

Fused in sarcoma (FUS) encodes an RNA-binding protein with diverse roles in transcriptional activation and RNA splicing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS can cause amyotrophic lateral sclerosis. FUS has also been implicated in genome maintenance. However, the underlying mechanisms of its actions in genome stability are unknown. Here, we applied gene editing, functional reconstitution, and integrated proteomics and transcriptomics to illuminate roles for FUS in DNA replication and repair. Consistent with a supportive role in DNA double-strand break repair, FUS-deficient cells exhibited subtle alterations in the recruitment and retention of double-strand break-associated factors, including 53BP1 and BRCA1. FUS-/- cells also exhibited reduced proliferative potential that correlated with reduced speed of replication fork progression, diminished loading of prereplication complexes, enhanced micronucleus formation, and attenuated expression and splicing of S-phase-associated genes. Finally, FUS-deficient cells exhibited genome-wide alterations in DNA replication timing that were reversed upon re-expression of FUS complementary DNA. We also showed that FUS-dependent replication domains were enriched in transcriptionally active chromatin and that FUS was required for the timely replication of transcriptionally active DNA. These findings suggest that alterations in DNA replication kinetics and programming contribute to genome instability and functional defects in FUS-deficient cells.

Co-opting the Fanconi anemia genomic stability pathway enables herpesvirus DNA synthesis and productive growth.

DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

Nuclear export of the NF-κB inhibitor IκBα is required for proper B cell and secondary lymphoid tissue formation.

The N-terminal nuclear export sequence (NES) of inhibitor of nuclear factor kappa B (NF-κB) alpha (IκBα) promotes NF-κB export from the cell nucleus to the cytoplasm, but the physiological role of this export regulation remains unknown. Here we report the derivation and analysis of genetically targeted mice harboring a germline mutation in IκBα NES. Mature B cells in the mutant mice displayed nuclear accumulation of inactive IκBα complexes containing a NF-κB family member, cRel, causing their spatial separation from the cytoplasmic IκB kinase. This resulted in severe reductions in constitutive and canonical NF-κB activities, synthesis of p100 and RelB NF-κB members, noncanonical NF-κB activity, NF-κB target gene induction, and proliferation and survival responses in B cells. Consequently, mice displayed defective B cell maturation, antibody production, and formation of secondary lymphoid organs and tissues. Thus, IκBα nuclear export is essential to maintain constitutive, canonical, and noncanonical NF-κB activation potentials in mature B cells in vivo.

The auto-generated fragment of the Usp1 deubiquitylase is a physiological substrate of the N-end rule pathway.

Deamidation of N-terminal Gln by the Ntaq1 Nt(Q)-amidase is a part of the Arg/N-end rule pathway, a ubiquitin-dependent proteolytic system. Here we identify Gln-Usp1(Ct), the C-terminal fragment of the autocleaved Usp1 deubiquitylase, as the first physiological Arg/N-end rule substrate that is targeted for degradation through deamidation of N-terminal Gln. Usp1 regulates genomic stability, in part through the deubiquitylation of monoubiquitylated PCNA, a DNA polymerase processivity factor. The autocleaved Usp1 remains a deubiquitylase because its fragments remain associated with Uaf1, an enhancer of Usp1 activity, until the Gln-Usp1(Ct) fragment is selectively destroyed by the Arg/N-end rule pathway. We also show that metabolic stabilization of Gln-Usp1(Ct) results in a decreased monoubiquitylation of PCNA and in a hypersensitivity of cells to ultraviolet irradiation. Thus, in addition to its other functions in DNA repair and chromosome segregation, the Arg/N-end rule pathway regulates genomic stability through the degradation-mediated control of the autocleaved Usp1 deubiquitylase.

Dysregulation of DNA polymerase κ recruitment to replication forks results in genomic instability.

Translesion synthesis polymerases (TLS Pols) are required to tolerate DNA lesions that would otherwise cause replication arrest and cell death. Aberrant expression of these specialized Pols may be responsible for increased mutagenesis and loss of genome integrity in human cancers. The molecular events that control the usage of TLS Pols in non-pathological conditions remain largely unknown. Here, we show that aberrant recruitment of TLS Polκ to replication forks results in genomic instability and can be mediated through the loss of the deubiquitinase USP1. Moreover, artificial tethering of Polκ to proliferating cell nuclear antigen (PCNA) circumvents the need for its ubiquitin-binding domain in the promotion of genomic instability. Finally, we show that the loss of USP1 leads to a dramatic reduction of replication fork speed in a Polκ-dependent manner. We propose a mechanism whereby reversible ubiquitination of PCNA can prevent spurious TLS Pol recruitment and regulate replication fork speed to ensure the maintenance of genome integrity.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair.

Fanconi anemia (FA) is a rare familial genome instability syndrome caused by mutations in FA genes that results in defective DNA crosslink repair. Activation of the FA pathway requires the FA core ubiquitin ligase complex-dependent monoubiquitination of 2 interacting FA proteins, FANCI and FANCD2. Although loss of either FANCI or FANCD2 is known to prevent monoubiquitination of its respective partner, it is unclear whether FANCI has any additional domains that may be important in promoting DNA repair, independent of its monoubiquitination. Here, we focus on an FA-I patient-derived FANCI mutant protein, R1299X (deletion of 30 residues from its C-terminus), to characterize important structural region(s) in FANCI that is required to activate the FA pathway. We show that, within this short 30 amino acid stretch contains 2 separable functional signatures, a nuclear localization signal and a putative EDGE motif, that is critical for the ability of FANCI to properly monoubiquitinate FANCD2 and promote DNA crosslink resistance. Our study enable us to conclude that, although proper nuclear localization of FANCI is crucial for robust FANCD2 monoubiquitination, the putative FANCI EDGE motif is important for DNA crosslink repair.

Regulation of monoubiquitinated PCNA by DUB autocleavage.

Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.

The Fanconi anemia pathway in replication stress and DNA crosslink repair.

Interstand crosslinks (ICLs) are DNA lesions where the bases of opposing DNA strands are covalently linked, inhibiting critical cellular processes such as transcription and replication. Chemical agents that generate ICLs cause chromosomal abnormalities including breaks, deletions and rearrangements, making them highly genotoxic compounds. This toxicity has proven useful for chemotherapeutic treatment against a wide variety of cancer types. The majority of our understanding of ICL repair in humans has been uncovered through analysis of the rare genetic disorder Fanconi anemia, in which patients are extremely sensitive to crosslinking agents. Here, we discuss recent insights into ICL repair gained using new repair assays and highlight the role of the Fanconi anemia repair pathway during replication stress.