Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.

Lesion Bypass and the Reactivation of Stalled Replication Forks.

Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. However, the idea that replication forks would form at origins of DNA replication and proceed without impairment to copy the chromosomes has proven naive. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes, unrelieved positive superhelical tension, covalent protein-DNA complexes, and as a result of cellular stress responses. These stalled forks are a major source of genome instability. The cell has developed many strategies for ensuring that these obstructions to DNA replication do not result in loss of genetic information, including DNA damage tolerance mechanisms such as lesion skipping, whereby the replisome jumps the lesion and continues downstream; template switching both behind template damage and at the stalled fork; and the error-prone pathway of translesion synthesis.

Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe.

Telomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1-/- cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres.

Integrator facilitates RNAPII removal to prevent transcription-replication collisions and genome instability.

DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.

DNA-PKcs promotes fork reversal and chemoresistance.

The DNA-PKcs kinase mediates the repair of DNA double-strand breaks via classical non-homologous end joining (NHEJ). DNA-PKcs is also recruited to active replication forks, although a role for DNA-PKcs in the control of fork dynamics is unclear. Here, we identify a crucial role for DNA-PKcs in promoting fork reversal, a process that stabilizes stressed replication forks and protects genome integrity. DNA-PKcs promotes fork reversal and slowing in response to several replication stress-inducing agents in a manner independent of its role in NHEJ. Cells lacking DNA-PKcs activity show increased DNA damage during S-phase and cellular sensitivity to replication stress. Notably, prevention of fork slowing and reversal via DNA-PKcs inhibition efficiently restores chemotherapy sensitivity in BRCA2-deficient mammary tumors with acquired PARPi resistance. Together, our data uncover a new key regulator of fork reversal and show how DNA-PKcs signaling can be manipulated to alter fork dynamics and drug resistance in cancer.

RADX controls RAD51 filament dynamics to regulate replication fork stability.

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication.

CARM1 regulates replication fork speed and stress response by stimulating PARP1.

DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms.

The ZATT-TOP2A-PICH Axis Drives Extensive Replication Fork Reversal to Promote Genome Stability.

Replication fork reversal is a global response to replication stress in mammalian cells, but precisely how it occurs remains poorly understood. Here, we show that, upon replication stress, DNA topoisomerase IIalpha (TOP2A) is recruited to stalled forks in a manner dependent on the SNF2-family DNA translocases HLTF, ZRANB3, and SMARCAL1. This is accompanied by an increase in TOP2A SUMOylation mediated by the SUMO E3 ligase ZATT and followed by recruitment of a SUMO-targeted DNA translocase, PICH. Disruption of the ZATT-TOP2A-PICH axis results in accumulation of partially reversed forks and enhanced genome instability. These results suggest that fork reversal occurs via a sequential two-step process. First, HLTF, ZRANB3, and SMARCAL1 initiate limited fork reversal, creating superhelical strain in the newly replicated sister chromatids. Second, TOP2A drives extensive fork reversal by resolving the resulting topological barriers and via its role in recruiting PICH to stalled forks.

MYC Controls the Epstein-Barr Virus Lytic Switch.

Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.

ATR Protects the Genome against R Loops through a MUS81-Triggered Feedback Loop.

R loops arising during transcription induce genomic instability, but how cells respond to the R loop-associated genomic stress is still poorly understood. Here, we show that cells harboring high levels of R loops rely on the ATR kinase for survival. In response to aberrant R loop accumulation, the ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway is activated by R loop-induced reversed replication forks. In contrast to the activation of ATR by replication inhibitors, R loop-induced ATR activation requires the MUS81 endonuclease. ATR protects the genome from R loops by suppressing transcription-replication collisions, promoting replication fork recovery, and enforcing a G2/M cell-cycle arrest. Furthermore, ATR prevents excessive cleavage of reversed forks by MUS81, revealing a MUS81-triggered and ATR-mediated feedback loop that fine-tunes MUS81 activity at replication forks. These results suggest that ATR is a key sensor and suppressor of R loop-induced genomic instability, uncovering a signaling circuitry that safeguards the genome against R loops.

PRIMPOL-Mediated Adaptive Response Suppresses Replication Fork Reversal in BRCA-Deficient Cells.

Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis.

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.

Oral Anticoagulants Beyond Warfarin.

Direct oral anticoagulants (DOACs) have largely replaced vitamin K antagonists, mostly warfarin, for the main indications for oral anticoagulation, prevention and treatment of venous thromboembolism, and prevention of embolic stroke in atrial fibrillation. While DOACs offer practical, fixed-dose anticoagulation in many patients, specific restrictions or contraindications may apply. DOACs are not sufficiently effective in high-thrombotic risk conditions such as antiphospholipid syndrome and mechanical heart valves. Patients with cancer-associated thrombosis may benefit from DOACs, but the bleeding risk, particularly in those with gastrointestinal or urogenital tumors, must be carefully weighed. In patients with frailty, excess body weight, and/or moderate-to-severe chronic kidney disease, DOACs must be cautiously administered and may require laboratory monitoring. Reversal agents have been developed and approved for life-threatening bleeding. In addition, the clinical testing of potentially safer anticoagulants such as factor XI(a) inhibitors is important to further optimize anticoagulant therapy in an increasingly elderly and frail population worldwide.

Replication-Coupled DNA Repair.

The replisome quickly and accurately copies billions of DNA bases each cell division cycle. However, it can make errors, especially when the template DNA is damaged. In these cases, replication-coupled repair mechanisms remove the mistake or repair the template lesions to ensure high fidelity and complete copying of the genome. Failures in these genome maintenance activities generate mutations, rearrangements, and chromosome segregation problems that cause many human diseases. In this review, I provide a broad overview of replication-coupled repair pathways, explaining how they fix polymerase mistakes, respond to template damage that acts as obstacles to the replisome, deal with broken forks, and impact human health and disease.

Development of an aptamer capable of multidrug resistance reversal for tumor combination chemotherapy.

Multidrug resistance (MDR) is a major factor in the failure of many forms of tumor chemotherapy. Development of a specific ligand for MDR-reversal would enhance the intracellular accumulation of therapeutic agents and effectively improve the tumor treatments. Here, an aptamer was screened against a doxorubicin (DOX)-resistant human hepatocellular carcinoma cell line (HepG2/DOX) via cell-based systematic evolution of ligands by exponential enrichment. A 50 nt truncated sequence termed d3 was obtained with high affinity and specificity for HepG2/DOX cells. Multidrug resistance protein 1 (MDR1) is determined to be a possible recognition target of the selected aptamer. Aptamer d3 binding was revealed to block the MDR of the tumor cells and increase the accumulation of intracellular anticancer drugs, including DOX, vincristine, and paclitaxel, which led to a boost to the cell killing of the anticancer drugs and lowering their survival of the tumor cells. The aptamer d3-mediated MDR-reversal for effective chemotherapy was further verified in an in vivo animal model, and combination of aptamer d3 with DOX significantly improved the suppression of tumor growth by treating a xenograft HepG2/DOX tumor in vivo. This work demonstrates the feasibility of a therapeutic DNA aptamer as a tumor MDR-reversal agent, and combination of the selected aptamer with chemotherapeutic drugs shows great potential for liver cancer treatments.

The evolution of sex roles: The importance of ecology and social environment.

Males and females often have different roles in reproduction, although the origin of these differences has remained controversial. Explaining the enigmatic reversed sex roles where males sacrifice their mating potential and provide full parental care is a particularly long-standing challenge in evolutionary biology. While most studies focused on ecological factors as the drivers of sex roles, recent research highlights the significance of social factors such as the adult sex ratio. To disentangle these propositions, here, we investigate the additive and interactive effects of several ecological and social factors on sex role variation using shorebirds (sandpipers, plovers, and allies) as model organisms that provide the full spectrum of sex role variation including some of the best-known examples of sex-role reversal. Our results consistently show that social factors play a prominent role in driving sex roles. Importantly, we show that reversed sex roles are associated with both male-skewed adult sex ratios and high breeding densities. Furthermore, phylogenetic path analyses provide general support for sex ratios driving sex role variations rather than being a consequence of sex roles. Together, these important results open future research directions by showing that different mating opportunities of males and females play a major role in generating the evolutionary diversity of sex roles, mating system, and parental care.

Fluctuating selection and the determinants of genetic variation.

Recent studies of cosmopolitan Drosophila populations have found hundreds to thousands of genetic loci with seasonally fluctuating allele frequencies, bringing temporally fluctuating selection to the forefront of the historical debate surrounding the maintenance of genetic variation in natural populations. Numerous mechanisms have been explored in this longstanding area of research, but these exciting empirical findings have prompted several recent theoretical and experimental studies that seek to better understand the drivers, dynamics, and genome-wide influence of fluctuating selection. In this review, we evaluate the latest evidence for multilocus fluctuating selection in Drosophila and other taxa, highlighting the role of potential genetic and ecological mechanisms in maintaining these loci and their impacts on neutral genetic variation.

New insights into transcription elongation control of HIV-1 latency and rebound.

Antiretroviral therapy reduces circulating HIV-1 to undetectable amounts but does not eliminate the virus due to the persistence of a stable reservoir of latently infected cells. The reservoir is maintained both by proliferation of latently infected cells and by reseeding from reactivated cells. A major challenge for the field is to find safe and effective methods to eliminate this source of rebounding HIV-1. Studies on the molecular mechanisms leading to HIV-1 latency and reactivation are being transformed using latency models in primary and patient CD4+ T cells. These studies have revealed the central role played by the biogenesis of the transcription elongation factor P-TEFb (Positive Transcription Elongation Factor b) and its recruitment to proviral HIV-1, for the maintenance of viral latency and the control of viral reactivation.

Histone Ubiquitination by the DNA Damage Response Is Required for Efficient DNA Replication in Unperturbed S Phase.

Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.