Journal articles: 'Fork restart'

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Gold, Michaela A., Jenna M. Whalen, Karine Freon, Zixin Hong, Ismail Iraqui, Sarah A. E. Lambert, and Catherine H. Freudenreich. "Restarted replication forks are error-prone and cause CAG repeat expansions and contractions." PLOS Genetics 17, no. 10 (October 21, 2021): e1009863. http://dx.doi.org/10.1371/journal.pgen.1009863.

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Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2–7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.

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Petermann, Eva, and Thomas Helleday. "Pathways of mammalian replication fork restart." Nature Reviews Molecular Cell Biology 11, no. 10 (September 15, 2010): 683–87. http://dx.doi.org/10.1038/nrm2974.

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Pepe, Alessandra, and Stephen C. West. "MUS81-EME2 Promotes Replication Fork Restart." Cell Reports 7, no. 4 (May 2014): 1048–55. http://dx.doi.org/10.1016/j.celrep.2014.04.007.

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Dyankova-Danovska, Teodora, Sonya Uzunova, Georgi Danovski, Rumen Stamatov, Petar-Bogomil Kanev, Aleksandar Atemin, Aneliya Ivanova, Radoslav Aleksandrov, and Stoyno Stoynov. "In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell." International Journal of Molecular Sciences 26, no. 2 (January 14, 2025): 667. https://doi.org/10.3390/ijms26020667.

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Replication forks encounter various impediments, which induce fork stalling and threaten genome stability, yet the precise dynamics of fork stalling and restart at the single-cell level remain elusive. Herein, we devise a live-cell microscopy-based approach to follow hydroxyurea-induced fork stalling and subsequent restart at 30 s resolution. We measure two distinct processes during fork stalling. One is rapid PCNA removal, which reflects the drop in DNA synthesis. The other is gradual RPA1 accumulation up to 2400 nt of ssDNA per fork despite an active intra-S checkpoint. Restoring the nucleotide pool enables a prompt restart without post-replicative ssDNA and a smooth cell cycle progression. ATR, but not ATM inhibition, accelerates hydroxyurea-induced RPA1 accumulation nine-fold, leading to RPA1 exhaustion within 20 min. Fork restart under ATR inhibition led to the persistence of ~600 nt ssDNA per fork after S-phase, which reached 2500 nt under ATR/ATM co-inhibition, with both scenarios leading to mitotic catastrophe. MRE11 inhibition had no effect on PCNA/RPA1 dynamics regardless of ATR activity. E3 ligase RAD18 was recruited at stalled replication forks in parallel to PCNA removal. Our results shed light on fork dynamics during nucleotide depletion and provide a valuable tool for interrogating the effects of replication stress-inducing anti-cancer agents.

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Longerich, S., and P. Sung. "Clearance of roadblocks in replication fork restart." Proceedings of the National Academy of Sciences 108, no. 34 (August 8, 2011): 13881–82. http://dx.doi.org/10.1073/pnas.1110698108.

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Iyer, Divya R., and Alan D. D’Andrea. "Fork restart: unloading FANCD2 to travel ahead." Molecular Cell 83, no. 20 (October 2023): 3590–92. http://dx.doi.org/10.1016/j.molcel.2023.09.027.

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Thangavel, Saravanabhavan, Matteo Berti, Maryna Levikova, Cosimo Pinto, Shivasankari Gomathinayagam, Marko Vujanovic, Ralph Zellweger, et al. "DNA2 drives processing and restart of reversed replication forks in human cells." Journal of Cell Biology 208, no. 5 (March 2, 2015): 545–62. http://dx.doi.org/10.1083/jcb.201406100.

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Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.

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Eksi, Sebnem Ece, and Joshua C. Saldivar. "Cohesin Is Out for Stalled Replication Fork Restart." Developmental Cell 52, no. 6 (March 2020): 675–76. http://dx.doi.org/10.1016/j.devcel.2020.03.001.

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Marians, Kenneth J. "PriA-directed replication fork restart in Escherichia coli." Trends in Biochemical Sciences 25, no. 4 (April 2000): 185–89. http://dx.doi.org/10.1016/s0968-0004(00)01565-6.

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Marians, Kenneth J. "Mechanisms of replication fork restart in Escherichia coli." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 359, no. 1441 (January 29, 2004): 71–77. http://dx.doi.org/10.1098/rstb.2003.1366.

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Replication of the genome is crucial for the accurate transmission of genetic information. It has become clear over the last decade that the orderly progression of replication forks in both prokaryotes and eukaryotes is disrupted with high frequency by encounters with various obstacles either on or in the template strands. Survival of the organism then becomes dependent on both removal of the obstruction and resumption of replication. This latter point is particularly important in bacteria, where the number of replication forks per genome is nominally only two. Replication restart in Escherichia coli is accomplished by the action of the restart primosomal proteins, which use both recombination intermediates and stalled replication forks as substrates for loading new replication forks. These reactions have been reconstituted with purified recombination and replication proteins.

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Xu, Michelle J., and Philip W. Jordan. "SMC5/6 Promotes Replication Fork Stability via Negative Regulation of the COP9 Signalosome." International Journal of Molecular Sciences 25, no. 2 (January 12, 2024): 952. http://dx.doi.org/10.3390/ijms25020952.

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It is widely accepted that DNA replication fork stalling is a common occurrence during cell proliferation, but there are robust mechanisms to alleviate this and ensure DNA replication is completed prior to chromosome segregation. The SMC5/6 complex has consistently been implicated in the maintenance of replication fork integrity. However, the essential role of the SMC5/6 complex during DNA replication in mammalian cells has not been elucidated. In this study, we investigate the molecular consequences of SMC5/6 loss at the replication fork in mouse embryonic stem cells (mESCs), employing the auxin-inducible degron (AID) system to deplete SMC5 acutely and reversibly in the defined cellular contexts of replication fork stall and restart. In SMC5-depleted cells, we identify a defect in the restart of stalled replication forks, underpinned by excess MRE11-mediated fork resection and a perturbed localization of fork protection factors to the stalled fork. Previously, we demonstrated a physical and functional interaction of SMC5/6 with the COP9 signalosome (CSN), a cullin deneddylase that enzymatically regulates cullin ring ligase (CRL) activity. Employing a combination of DNA fiber techniques, the AID system, small-molecule inhibition assays, and immunofluorescence microscopy analyses, we show that SMC5/6 promotes the localization of fork protection factors to stalled replication forks by negatively modulating the COP9 signalosome (CSN). We propose that the SMC5/6-mediated modulation of the CSN ensures that CRL activity and their roles in DNA replication fork stabilization are maintained to allow for efficient replication fork restart when a replication fork stall is alleviated.

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Torres, Jorge Z., Sandra L. Schnakenberg, and Virginia A. Zakian. "Saccharomyces cerevisiae Rrm3p DNA Helicase Promotes Genome Integrity by Preventing Replication Fork Stalling: Viability of rrm3 Cells Requires the Intra-S-Phase Checkpoint and Fork Restart Activities." Molecular and Cellular Biology 24, no. 8 (April 15, 2004): 3198–212. http://dx.doi.org/10.1128/mcb.24.8.3198-3212.2004.

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ABSTRACT Rrm3p is a 5′-to-3′ DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.

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Bianco, Piero R., and Yue Lu. "Single-molecule insight into stalled replication fork rescue in Escherichia coli." Nucleic Acids Research 49, no. 8 (March 21, 2021): 4220–38. http://dx.doi.org/10.1093/nar/gkab142.

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Abstract DNA replication forks stall at least once per cell cycle in Escherichia coli. DNA replication must be restarted if the cell is to survive. Restart is a multi-step process requiring the sequential action of several proteins whose actions are dictated by the nature of the impediment to fork progression. When fork progress is impeded, the sequential actions of SSB, RecG and the RuvABC complex are required for rescue. In contrast, when a template discontinuity results in the forked DNA breaking apart, the actions of the RecBCD pathway enzymes are required to resurrect the fork so that replication can resume. In this review, we focus primarily on the significant insight gained from single-molecule studies of individual proteins, protein complexes, and also, partially reconstituted regression and RecBCD pathways. This insight is related to the bulk-phase biochemical data to provide a comprehensive review of each protein or protein complex as it relates to stalled DNA replication fork rescue.

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Jain, Chetan K., Swagata Mukhopadhyay, and Agneyo Ganguly. "RecQ Family Helicases in Replication Fork Remodeling and Repair: Opening New Avenues towards the Identification of Potential Targets for Cancer Chemotherapy." Anti-Cancer Agents in Medicinal Chemistry 20, no. 11 (July 8, 2020): 1311–26. http://dx.doi.org/10.2174/1871520620666200518082433.

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Replication fork reversal and restart has gained immense interest as a central response mechanism to replication stress following DNA damage. Although the exact mechanism of fork reversal has not been elucidated precisely, the involvement of diverse pathways and different factors has been demonstrated, which are central to this phenomenon. RecQ helicases known for their vital role in DNA repair and maintaining genome stability has recently been implicated in the restart of regressed replication forks. Through interaction with vital proteins like Poly (ADP) ribose polymerase 1 (PARP1), these helicases participate in the replication fork reversal and restart phenomenon. Most therapeutic agents used for cancer chemotherapy act by causing DNA damage in replicating cells and subsequent cell death. These DNA damages can be repaired by mechanisms involving fork reversal as the key phenomenon eventually reducing the efficacy of the therapeutic agent. Hence the factors contributing to this repair process can be good selective targets for developing more efficient chemotherapeutic agents. In this review, we have discussed in detail the role of various proteins in replication fork reversal and restart with special emphasis on RecQ helicases. Involvement of other proteins like PARP1, recombinase rad51, SWI/SNF complex has also been discussed. Since RecQ helicases play a central role in the DNA damage response following chemotherapeutic treatment, we propose that targeting these helicases can emerge as an alternative to available intervention strategies. We have also summarized the current research status of available RecQ inhibitors and siRNA based therapeutic approaches that targets RecQ helicases. In summary, our review gives an overview of the DNA damage responses involving replication fork reversal and provides new directions for the development of more efficient and sustainable chemotherapeutic approaches.

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Grompone, Gianfranco, Dusko Ehrlich, and Bénédicte Michel. "Cells defective for replication restart undergo replication fork reversal." EMBO reports 5, no. 6 (May 28, 2004): 607–12. http://dx.doi.org/10.1038/sj.embor.7400167.

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Manosas, M., S. K. Perumal, V. Croquette, and S. J. Benkovic. "Direct Observation of Stalled Fork Restart via Fork Regression in the T4 Replication System." Science 338, no. 6111 (November 29, 2012): 1217–20. http://dx.doi.org/10.1126/science.1225437.

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Yates, Maïlyn, and Alexandre Maréchal. "Ubiquitylation at the Fork: Making and Breaking Chains to Complete DNA Replication." International Journal of Molecular Sciences 19, no. 10 (September 25, 2018): 2909. http://dx.doi.org/10.3390/ijms19102909.

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The complete and accurate replication of the genome is a crucial aspect of cell proliferation that is often perturbed during oncogenesis. Replication stress arising from a variety of obstacles to replication fork progression and processivity is an important contributor to genome destabilization. Accordingly, cells mount a complex response to this stress that allows the stabilization and restart of stalled replication forks and enables the full duplication of the genetic material. This response articulates itself on three important platforms, Replication Protein A/RPA-coated single-stranded DNA, the DNA polymerase processivity clamp PCNA and the FANCD2/I Fanconi Anemia complex. On these platforms, the recruitment, activation and release of a variety of genome maintenance factors is regulated by post-translational modifications including mono- and poly-ubiquitylation. Here, we review recent insights into the control of replication fork stability and restart by the ubiquitin system during replication stress with a particular focus on human cells. We highlight the roles of E3 ubiquitin ligases, ubiquitin readers and deubiquitylases that provide the required flexibility at stalled forks to select the optimal restart pathways and rescue genome stability during stressful conditions.

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Leuzzi, Giuseppe, Veronica Marabitti, Pietro Pichierri, and Annapaola Franchitto. "WRNIP 1 protects stalled forks from degradation and promotes fork restart after replication stress." EMBO Journal 35, no. 13 (May 30, 2016): 1437–51. http://dx.doi.org/10.15252/embj.201593265.

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Polleys, Erica J., Nealia C. M. House, and Catherine H. Freudenreich. "Role of recombination and replication fork restart in repeat instability." DNA Repair 56 (August 2017): 156–65. http://dx.doi.org/10.1016/j.dnarep.2017.06.018.

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Raghunandan, Maya, Jung Eun Yeo, Ryan Walter, Kai Saito, Adam J. Harvey, Stacie Ittershagen, Eun-A. Lee, et al. "Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery." Human Molecular Genetics 29, no. 7 (October 19, 2019): 1083–95. http://dx.doi.org/10.1093/hmg/ddz250.

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Abstract Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase.

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Batenburg, Nicole L., Sofiane Y. Mersaoui, John R. Walker, Yan Coulombe, Ian Hammond-Martel, Hugo Wurtele, Jean-Yves Masson, and Xu-Dong Zhu. "Cockayne syndrome group B protein regulates fork restart, fork progression and MRE11-dependent fork degradation in BRCA1/2-deficient cells." Nucleic Acids Research 49, no. 22 (December 6, 2021): 12836–54. http://dx.doi.org/10.1093/nar/gkab1173.

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Abstract Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.

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Feu, Sonia, Fernando Unzueta, Amaia Ercilla, Alejandro Pérez-Venteo, Montserrat Jaumot, and Neus Agell. "RAD51 is a druggable target that sustains replication fork progression upon DNA replication stress." PLOS ONE 17, no. 8 (August 15, 2022): e0266645. http://dx.doi.org/10.1371/journal.pone.0266645.

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Solving the problems that replication forks encounter when synthesizing DNA is essential to prevent genomic instability. Besides their role in DNA repair in the G2 phase, several homologous recombination proteins, specifically RAD51, have prominent roles in the S phase. Using different cellular models, RAD51 has been shown not only to be present at ongoing and arrested replication forks but also to be involved in nascent DNA protection and replication fork restart. Through pharmacological inhibition, here we study the specific role of RAD51 in the S phase. RAD51 inhibition in non-transformed cell lines did not have a significant effect on replication fork progression under non-perturbed conditions, but when the same cells were subjected to replication stress, RAD51 became necessary to maintain replication fork progression. Notably, the inhibition or depletion of RAD51 did not compromise fork integrity when subjected to hydroxyurea treatment. RAD51 inhibition also did not decrease the ability to restart, but rather compromised fork progression during reinitiation. In agreement with the presence of basal replication stress in human colorectal cancer cells, RAD51 inhibition reduced replication fork speed in these cells and increased γH2Ax foci under control conditions. These alterations could have resulted from the reduced association of DNA polymerase α to chromatin, as observed when inhibiting RAD51. It may be possible to exploit the differential dependence of non-transformed cells versus colorectal cancer cells on RAD51 activity under basal conditions to design new therapies that specifically target cancer cells.

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Liu, Wenpeng, Yuichiro Saito, Jessica Jackson, Rahul Bhowmick, Masato T. Kanemaki, Alessandro Vindigni, and David Cortez. "RAD51 bypasses the CMG helicase to promote replication fork reversal." Science 380, no. 6643 (April 28, 2023): 382–87. http://dx.doi.org/10.1126/science.add7328.

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Replication fork reversal safeguards genome integrity as a replication stress response. DNA translocases and the RAD51 recombinase catalyze reversal. However, it remains unknown why RAD51 is required and what happens to the replication machinery during reversal. We find that RAD51 uses its strand exchange activity to circumvent the replicative helicase, which remains bound to the stalled fork. RAD51 is not required for fork reversal if the helicase is unloaded. Thus, we propose that RAD51 creates a parental DNA duplex behind the helicase that is used as a substrate by the DNA translocases for branch migration to create a reversed fork structure. Our data explain how fork reversal happens while maintaining the helicase in a position poised to restart DNA synthesis and complete genome duplication.

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Miyabe, Izumi, Ken'Ichi Mizuno, Andrea Keszthelyi, Yasukazu Daigaku, Meliti Skouteri, Saed Mohebi, Thomas A. Kunkel, Johanne M. Murray, and Antony M. Carr. "Polymerase δ replicates both strands after homologous recombination–dependent fork restart." Nature Structural & Molecular Biology 22, no. 11 (October 5, 2015): 932–38. http://dx.doi.org/10.1038/nsmb.3100.

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Szyjka, S. J., J. G. Aparicio, C. J. Viggiani, S. Knott, W. Xu, S. Tavare, and O. M. Aparicio. "Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae." Genes & Development 22, no. 14 (July 15, 2008): 1906–20. http://dx.doi.org/10.1101/gad.1660408.

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Croquette, Vincent, Maria Manosas, Senthil K. Perumal, and Stephen J. Benkovic. "Direct Observation of Stalled Fork Restart and Lesion Bypass via Fork Regression in the T4 Replication System." Biophysical Journal 104, no. 2 (January 2013): 367a—368a. http://dx.doi.org/10.1016/j.bpj.2012.11.2042.

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Lo, Calvin Shun Yu, Marvin van Toorn, Vincent Gaggioli, Mariana Paes Dias, Yifan Zhu, Eleni Maria Manolika, Wei Zhao, et al. "SMARCAD1-mediated active replication fork stability maintains genome integrity." Science Advances 7, no. 19 (May 2021): eabe7804. http://dx.doi.org/10.1126/sciadv.abe7804.

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The stalled fork protection pathway mediated by breast cancer 1/2 (BRCA1/2) proteins is critical for replication fork stability. However, it is unclear whether additional mechanisms are required to maintain replication fork stability. We describe a hitherto unknown mechanism, by which the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily-A containing DEAD/H box-1 (SMARCAD1) stabilizes active replication forks, that is essential to maintaining resistance towards replication poisons. We find that SMARCAD1 prevents accumulation of 53BP1-associated nucleosomes to preclude toxic enrichment of 53BP1 at the forks. In the absence of SMARCAD1, 53BP1 mediates untimely dissociation of PCNA via the PCNA-unloader ATAD5, causing frequent fork stalling, inefficient fork restart, and accumulation of single-stranded DNA. Although loss of 53BP1 in SMARCAD1 mutants rescues these defects and restores genome stability, this rescued stabilization also requires BRCA1-mediated fork protection. Notably, fork protection-challenged BRCA1-deficient naïve- or chemoresistant tumors require SMARCAD1-mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

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Schwab, Rebekka A., Jadwiga Nieminuszczy, Kazuo Shin-ya, and Wojciech Niedzwiedz. "FANCJ couples replication past natural fork barriers with maintenance of chromatin structure." Journal of Cell Biology 201, no. 1 (March 25, 2013): 33–48. http://dx.doi.org/10.1083/jcb.201208009.

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Defective DNA repair causes Fanconi anemia (FA), a rare childhood cancer–predisposing syndrome. At least 15 genes are known to be mutated in FA; however, their role in DNA repair remains unclear. Here, we show that the FANCJ helicase promotes DNA replication in trans by counteracting fork stalling on replication barriers, such as G4 quadruplex structures. Accordingly, stabilization of G4 quadruplexes in ΔFANCJ cells restricts fork movements, uncouples leading- and lagging-strand synthesis and generates small single-stranded DNA gaps behind the fork. Unexpectedly, we also discovered that FANCJ suppresses heterochromatin spreading by coupling fork movement through replication barriers with maintenance of chromatin structure. We propose that FANCJ plays an essential role in counteracting chromatin compaction associated with unscheduled replication fork stalling and restart, and suppresses tumorigenesis, at least partially, in this replication-specific manner.

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Thakur, Varsha, Juliano Tiburcio de Freitas, Yuan Li, Keman Zhang, Alyssa Savadelis, and Barbara Bedogni. "MT1-MMP-dependent ECM processing regulates laminB1 stability and mediates replication fork restart." PLOS ONE 16, no. 7 (July 8, 2021): e0253062. http://dx.doi.org/10.1371/journal.pone.0253062.

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Radiotherapy remains a mainstay of treatment for a majority of cancer patients. We have previously shown that the membrane bound matrix metalloproteinase MT1-MMP confers radio- and chemotherapy resistance to breast cancer via processing of the ECM and activation of integrinβ1/FAK signaling. Here, we further discovered that the nuclear envelope protein laminB1 is a potential target of integrinβ1/FAK. FAK interacts with laminB1 contributing to its stability. Stable laminB1 is found at replication forks (RFs) where it is likely to allow the proper positioning of RF protection factors, thus preventing RF degradation. Indeed, restoration of laminB1 expression rescues replication fork stalling and collapse that occurs upon MT1-MMP inhibition, and reduces DNA damage in breast cancer cells. Together, these data highlight a novel mechanism of laminB1 stability and replication fork restart via MT1-MMP dependent extracelluar matrix remodeling.

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Batenburg, Nicole L., John R. Walker, and Xu-Dong Zhu. "CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin." International Journal of Molecular Sciences 24, no. 15 (August 4, 2023): 12419. http://dx.doi.org/10.3390/ijms241512419.

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Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response to a low dose of CPT. At a high concentration of CPT, CSB is required to promote the restart of DNA replication through MUS81–RAD52–POLD3-dependent break-induced replication (BIR). In the absence of CSB, resumption of DNA synthesis at a high concentration of CPT can occur through POLQ–LIG3-, LIG4-, or PRIMPOL-dependent pathways, which are inhibited, respectively, by RAD51, BRCA1, and BRCA2 proteins. POLQ and LIG3 are core components of alternative end joining (Alt-EJ), whereas LIG4 is a core component of nonhomologous end joining (NHEJ). These results suggest that CSB regulates fork restart pathway choice following high-dosage CPT-induced fork stalling, promoting BIR but inhibiting Alt-EJ, NHEJ, and fork repriming. We find that loss of CSB and BRCA2 is a toxic combination to genomic stability and cell survival at a high concentration of CPT, which is likely due to accumulation of ssDNA gaps, underscoring an important role of CSB in regulating the therapy response in cancers lacking functional BRCA2.

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Chappidi, Nagaraja, Zuzana Nascakova, Barbora Boleslavska, Ralph Zellweger, Esin Isik, Martin Andrs, Shruti Menon, et al. "Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops." Molecular Cell 77, no. 3 (February 2020): 528–41. http://dx.doi.org/10.1016/j.molcel.2019.10.026.

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Hromas, R., E. A. Williamson, S. Fnu, Y.-J. Lee, S.-J. Park, B. D. Beck, J.-S. You, A. Laitao, J. A. Nickoloff, and S.-H. Lee. "Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart." Oncogene 31, no. 38 (January 9, 2012): 4245–54. http://dx.doi.org/10.1038/onc.2011.586.

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Schwab, Rebekka A., Andrew N. Blackford, and Wojciech Niedzwiedz. "ATR activation and replication fork restart are defective in FANCM-deficient cells." EMBO Journal 29, no. 4 (January 7, 2010): 806–18. http://dx.doi.org/10.1038/emboj.2009.385.

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Tittel-Elmer, Mireille, Armelle Lengronne, Marta B. Davidson, Julien Bacal, Philippe François, Marcel Hohl, John H. J. Petrini, Philippe Pasero, and Jennifer A. Cobb. "Cohesin Association to Replication Sites Depends on Rad50 and Promotes Fork Restart." Molecular Cell 48, no. 1 (October 2012): 98–108. http://dx.doi.org/10.1016/j.molcel.2012.07.004.

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35

Wang, Yaqing, Zhiqiang Sun, Piero R. Bianco, and Yuri L. Lyubchenko. "Atomic force microscopy–based characterization of the interaction of PriA helicase with stalled DNA replication forks." Journal of Biological Chemistry 295, no. 18 (March 24, 2020): 6043–52. http://dx.doi.org/10.1074/jbc.ra120.013013.

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In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3′-to-5′ direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA–binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5′- and 3′-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

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Bolt, E. L. "Helicases that interact with replication forks: new candidates from archaea." Biochemical Society Transactions 33, no. 6 (October 26, 2005): 1471–73. http://dx.doi.org/10.1042/bst0331471.

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Overcoming DNA replication fork blocks is essential for completing genome duplication and cell division. Archaea and eukaryotes drive replication using essentially the same protein machinery. Archaea may be a valuable resource for identifying new helicase components at advancing forks and/or in replication-restart pathways. As described here, these may be relevant to understanding genome instability in metazoans.

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Zellweger, Ralph, Damian Dalcher, Karun Mutreja, Matteo Berti, Jonas A. Schmid, Raquel Herrador, Alessandro Vindigni, and Massimo Lopes. "Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells." Journal of Cell Biology 208, no. 5 (March 2, 2015): 563–79. http://dx.doi.org/10.1083/jcb.201406099.

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Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy.

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Bianco, Piero R. "DNA Helicase-SSB Interactions Critical to the Regression and Restart of Stalled DNA Replication Forks in Escherichia coli." Genes 11, no. 5 (April 26, 2020): 471. http://dx.doi.org/10.3390/genes11050471.

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In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

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Nickoloff, Jac A., Neelam Sharma, Lynn Taylor, Sage J. Allen, and Robert Hromas. "Nucleases and Co-Factors in DNA Replication Stress Responses." DNA 2, no. 1 (March 1, 2022): 68–85. http://dx.doi.org/10.3390/dna2010006.

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DNA replication stress is a constant threat that cells must manage to proliferate and maintain genome integrity. DNA replication stress responses, a subset of the broader DNA damage response (DDR), operate when the DNA replication machinery (replisome) is blocked or replication forks collapse during S phase. There are many sources of replication stress, such as DNA lesions caused by endogenous and exogenous agents including commonly used cancer therapeutics, and difficult-to-replicate DNA sequences comprising fragile sites, G-quadraplex DNA, hairpins at trinucleotide repeats, and telomeres. Replication stress is also a consequence of conflicts between opposing transcription and replication, and oncogenic stress which dysregulates replication origin firing and fork progression. Cells initially respond to replication stress by protecting blocked replisomes, but if the offending problem (e.g., DNA damage) is not bypassed or resolved in a timely manner, forks may be cleaved by nucleases, inducing a DNA double-strand break (DSB) and providing a means to accurately restart stalled forks via homologous recombination. However, DSBs pose their own risks to genome stability if left unrepaired or misrepaired. Here we focus on replication stress response systems, comprising DDR signaling, fork protection, and fork processing by nucleases that promote fork repair and restart. Replication stress nucleases include MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, and FEN1. Replication stress factors are important in cancer etiology as suppressors of genome instability associated with oncogenic mutations, and as potential cancer therapy targets to enhance the efficacy of chemo- and radiotherapeutics.

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Singh, Mayank, Clayton R. Hunt, Raj K. Pandita, Rakesh Kumar, Chin-Rang Yang, Nobuo Horikoshi, Robert Bachoo, et al. "Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest." Molecular and Cellular Biology 33, no. 6 (January 14, 2013): 1210–22. http://dx.doi.org/10.1128/mcb.01676-12.

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The humanLMNAgene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations inLMNAresult in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.

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Tanaka, Taku, Yasumasa Nishito, and Hisao Masai. "Fork restart protein, PriA, binds around oriC after depletion of nucleotide precursors: Replication fork arrest near the replication origin." Biochemical and Biophysical Research Communications 470, no. 3 (February 2016): 546–51. http://dx.doi.org/10.1016/j.bbrc.2016.01.108.

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Bainbridge, Lewis J., Rebecca Teague, and Aidan J. Doherty. "Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication." Nucleic Acids Research 49, no. 9 (March 21, 2021): 4831–47. http://dx.doi.org/10.1093/nar/gkab176.

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Abstract To bypass a diverse range of fork stalling impediments encountered during genome replication, cells possess a variety of DNA damage tolerance (DDT) mechanisms including translesion synthesis, template switching, and fork reversal. These pathways function to bypass obstacles and allow efficient DNA synthesis to be maintained. In addition, lagging strand obstacles can also be circumvented by downstream priming during Okazaki fragment generation, leaving gaps to be filled post-replication. Whether repriming occurs on the leading strand has been intensely debated over the past half-century. Early studies indicated that both DNA strands were synthesised discontinuously. Although later studies suggested that leading strand synthesis was continuous, leading to the preferred semi-discontinuous replication model. However, more recently it has been established that replicative primases can perform leading strand repriming in prokaryotes. An analogous fork restart mechanism has also been identified in most eukaryotes, which possess a specialist primase called PrimPol that conducts repriming downstream of stalling lesions and structures. PrimPol also plays a more general role in maintaining efficient fork progression. Here, we review and discuss the historical evidence and recent discoveries that substantiate repriming as an intrinsic replication restart pathway for maintaining efficient genome duplication across all domains of life.

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Patel, Darshil R., and Robert S. Weiss. "A tough row to hoe: when replication forks encounter DNA damage." Biochemical Society Transactions 46, no. 6 (December 4, 2018): 1643–51. http://dx.doi.org/10.1042/bst20180308.

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Eukaryotic cells continuously experience DNA damage that can perturb key molecular processes like DNA replication. DNA replication forks that encounter DNA lesions typically slow and may stall, which can lead to highly detrimental fork collapse if appropriate protective measures are not executed. Stabilization and protection of stalled replication forks ensures the possibility of effective fork restart and prevents genomic instability. Recent efforts from multiple laboratories have highlighted several proteins involved in replication fork remodeling and DNA damage response pathways as key regulators of fork stability. Homologous recombination factors such as RAD51, BRCA1, and BRCA2, along with components of the Fanconi Anemia pathway, are now known to be crucial for stabilizing stalled replication forks and preventing nascent strand degradation. Several checkpoint proteins have additionally been implicated in fork protection. Ongoing work in this area continues to shed light on a sophisticated molecular pathway that balances the action of DNA resection and fork protection to maintain genomic integrity, with important implications for the fate of both normal and malignant cells following replication stress.

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Batté, Amandine, Sophie C. van der Horst, Mireille Tittel-Elmer, Su Ming Sun, Sushma Sharma, Jolanda van Leeuwen, Andrei Chabes, and Haico van Attikum. "Chl1 helicase controls replication fork progression by regulating dNTP pools." Life Science Alliance 5, no. 4 (January 11, 2022): e202101153. http://dx.doi.org/10.26508/lsa.202101153.

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Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

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45

Hromas, R., E. A. Williamson, S. Fnu, Y.-J. Lee, S.-J. Park, B. D. Beck, J.-S. You, A. Leitao, J. A. Nickoloff, and S.-H. Lee. "Erratum: Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart." Oncogene 33, no. 4 (January 2014): 536. http://dx.doi.org/10.1038/onc.2013.510.

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46

Stewart, Jason A., Feng Wang, Mary F. Chaiken, Christopher Kasbek, Paul D. Chastain, Woodring E. Wright, and Carolyn M. Price. "Human CST promotes telomere duplex replication and general replication restart after fork stalling." EMBO Journal 31, no. 17 (August 3, 2012): 3537–49. http://dx.doi.org/10.1038/emboj.2012.215.

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47

Pomerantz, R. T., and M. O'Donnell. "Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase." Science 327, no. 5965 (January 28, 2010): 590–92. http://dx.doi.org/10.1126/science.1179595.

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48

Jones, Rebecca M., and Eva Petermann. "Replication fork dynamics and the DNA damage response." Biochemical Journal 443, no. 1 (March 14, 2012): 13–26. http://dx.doi.org/10.1042/bj20112100.

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Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

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Lee, Han-Sae, Hye-Ran Seo, Shin-Ai Lee, Soohee Choi, Dongmin Kang, and Jongbum Kwon. "BAP1 promotes stalled fork restart and cell survival via INO80 in response to replication stress." Biochemical Journal 476, no. 20 (October 28, 2019): 3053–66. http://dx.doi.org/10.1042/bcj20190622.

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Abstract The recovery from replication stress by restarting stalled forks to continue DNA synthesis is crucial for maintaining genome stability and thereby preventing diseases such as cancer. We previously showed that BRCA1-associated protein 1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, promotes replication fork progression by stabilizing the INO80 chromatin remodeler via deubiquitination and recruiting it to replication forks during normal DNA synthesis. However, whether BAP1 functions in DNA replication under stress conditions is unknown. Here, we show that BAP1 depletion reduces S-phase progression and DNA synthesis after treatment with hydroxyurea (HU). BAP1-depleted cells exhibit a defect in the restart of HU-induced stalled replication forks, which is recovered by the ectopic expression of INO80. Both BAP1 and INO80 bind chromatin at replication forks upon HU treatment. BAP1 depletion abrogates the binding of INO80 to replication forks and increases the formation of RAD51 foci following HU treatment. BAP1-depleted cells show hypersensitivity to HU treatment, which is rescued by INO80 expression. These results suggest that BAP1 promotes the restart of stress-induced stalled replication forks by recruiting INO80 to the stalled forks. This function of BAP1 in replication stress recovery may contribute to its ability to suppress genome instability and cancer development.

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Yates, Maïlyn, Isabelle Marois, Edlie St-Hilaire, Daryl A. Ronato, Billel Djerir, Chloé Brochu, Théo Morin, et al. "SMARCAL1 ubiquitylation controls its association with RPA-coated ssDNA and promotes replication fork stability." PLOS Biology 22, no. 3 (March 19, 2024): e3002552. http://dx.doi.org/10.1371/journal.pbio.3002552.

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Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.

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Journal articles on the topic 'Fork restart'

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