scholarly journals A Critical Role of the 3′ Terminus of Nascent DNA Chains in Recognition of Stalled Replication Forks

2003 ◽  
Vol 278 (43) ◽  
pp. 42234-42239 ◽  
Author(s):  
Toshimi Mizukoshi ◽  
Taku Tanaka ◽  
Ken-ichi Arai ◽  
Daisuke Kohda ◽  
Hisao Masai
Keyword(s):  
Critical Role ◽  
Replication Forks ◽  
Stalled Replication Forks

scholarly journals The epigenetic regulator LSH maintains fork protection and genomic stability via MacroH2A deposition and RAD51 filament formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaoping Xu ◽  
Kai Ni ◽  
Yafeng He ◽  
Jianke Ren ◽  
Chongkui Sun ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Critical Role ◽  
Genomic Stability ◽  
Dna Degradation ◽  
Replication Forks ◽  
Filament Formation ◽  
Epigenetic Regulator ◽  
Centromeric Instability ◽  
Stalled Replication Forks

AbstractThe Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.

scholarly journals Regulation of Rtt107 Recruitment to Stalled DNA Replication Forks by the Cullin Rtt101 and the Rtt109 Acetyltransferase

2008 ◽  
Vol 19 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Tania M. Roberts ◽  
Iram Waris Zaidi ◽  
Jessica A. Vaisica ◽  
Matthias Peter ◽  
Grant W. Brown
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Replication Forks ◽  
Chromatin Binding ◽  
Direct Role ◽  
Repair Enzymes ◽  
Damage Response ◽  
Stalled Replication Forks

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint kinase Mec1, and it forms complexes with DNA repair enzymes, including the nuclease subunit Slx4, but the role of Rtt107 in the DNA damage response remains unclear. We find that Rtt107 interacts with chromatin when cells are treated with compounds that cause replication forks to arrest. This damage-dependent chromatin binding requires the acetyltransferase Rtt109, but it does not require acetylation of the known Rtt109 target, histone H3-K56. Chromatin binding of Rtt107 also requires the cullin Rtt101, which seems to play a direct role in Rtt107 recruitment, because the two proteins are found in complex with each other. Finally, we provide evidence that Rtt107 is bound at or near stalled replication forks in vivo. Together, these results indicate that Rtt109, Rtt101, and Rtt107, which genetic evidence suggests are functionally related, form a DNA damage response pathway that recruits Rtt107 complexes to damaged or stalled replication forks.

scholarly journals RAD51 and mitotic function of mus81 are essential for recovery from low-dose of camptothecin in the absence of the WRN exonuclease

2019 ◽  
Vol 47 (13) ◽  
pp. 6796-6810 ◽  
Author(s):  
Francesca Antonella Aiello ◽  
Anita Palma ◽  
Eva Malacaria ◽  
Li Zheng ◽  
Judith L Campbell ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Genome Instability ◽  
Genome Integrity ◽  
Replication Forks ◽  
Wild Type ◽  
Alternative Processing ◽  
Mitotic Abnormalities ◽  
Mitotic Phosphorylation ◽  
Stalled Replication Forks

Abstract Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.

scholarly journals Growth Phase Variation in Cell and Nucleoid Morphology in a Bacillus subtilis recAMutant

2001 ◽  
Vol 183 (9) ◽  
pp. 2963-2968 ◽  
Author(s):  
Stephen A. Sciochetti ◽  
Garry W. Blakely ◽  
Patrick J. Piggot
Keyword(s):  
Bacillus Subtilis ◽  
Growth Phase ◽  
Exponential Growth ◽  
Parent Strain ◽  
Phase Variation ◽  
Normal Growth ◽  
Replication Forks ◽  
Diminished Capacity ◽  
Stalled Replication Forks

ABSTRACT The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth,Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB andrecF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (∼15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition,recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.

scholarly journals Dimerization of the ATRIP Protein through the Coiled-Coil Motif and Its Implication to the Maintenance of Stalled Replication Forks

2005 ◽  
Vol 16 (12) ◽  
pp. 5551-5562 ◽  
Author(s):  
Eisuke Itakura ◽  
Isao Sawada ◽  
Akira Matsuura
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Genotoxic Stress ◽  
Replication Forks ◽  
Cycle Arrest ◽  
Coiled Coil Domain ◽  
Functional Complex ◽  
Stalled Replication Forks

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

The Recql5 Helicase Is a Novel Downstream Target of Jak2V617F and Maintains Genome Stability in Response to Replication Stress

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 822-822
Author(s):  
Edwin Chen ◽  
Jong-Sook Ahn ◽  
Lawrence J Breyfogle ◽  
Anthony R Green ◽  
Benjamin L. Ebert ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Myeloproliferative Neoplasms ◽  
Critical Role ◽  
Replication Stress ◽  
Therapeutic Window ◽  
Replication Forks ◽  
Erythroid Progenitor ◽  
Stalled Replication Forks

Abstract The JAK2V617F mutation is present in a majority of patients with chronic myeloproliferative neoplasms (MPNs). Mutant JAK2 induces hyperactivation of multiple downstream signaling processes with the net effect of conferring cells with a pro-survival advantage. In particular, JAK2V617F-expressing cells tolerate increased DNA damage and higher levels of intracellular reactive oxygen species. However, the mechanisms by which increased genotoxic tolerance is mediated remain unclear. Previously, we performed gene expression analysis on autologous wildtype and JAK2V617F-heterozygous erythroblasts from 36 MPN patients, and observed increased expression of the RECQL5 helicase in JAK2-mutant erythroblasts. Increased Recql5 transcript and protein levels were also validated in Hoxb8-immortalized, GMP-like cell lines derived from wildtype and Jak2V617F knock-in mice (WT-B8 and VF-B8 cells, respectively). Recql5 up-regulation was dependent on the Pi3k-Akt pathway, and was independent of Stat1/5 and Mapk/Erk activity. As the Recql family of helicases plays a critical role in replication fork stability, we tested whether Recql5 could modulate sensitivity of JAK2-expressing cells to agents which promote replication stress, such as hydroxyurea (HU) and aphidicolin (APH). Strikingly, VF-B8 cells transduced with two different Recql5 shRNAs were more susceptible to HU- and APH-induced apoptosis when compared to Recql5-deficient WT-B8 cells. Replication stress-induced cytotoxicity was accompanied by increased gamma-H2Ax-marked double stranded breaks (DSBs) and activation of DNA repair pathways. Importantly, re-introduction of an shRNA-resistant Recql5 cDNA successfully rescued Recql5-deficient VF-B8 cells from HU- and APH-cytotoxicity. Molecularly, we show that Recql5 plays two roles to protect against DSB formation and cell death in Jak2-mutant cells. First, we visualized replication tracts on individual DNA fibers by chromosome combing and observed that Recql5-deficient VF-B8 cells treated with HU exhibit increased numbers of stalled replication forks. Moreover, Recql5 deficiency also led to an inability to restart forks stalled by HU treatment. This indicates that the absence of Recql5 leads to replication forks which are unstably arrested upon HU treatment, leading to fork collapse and the generation of DSBs. Second, we quantified the rate of single-stranded annealing (SSA) repair following Recql5 knockdown. Consistent with previous reports, we observed increased rates of SSA repair in VF-B8 cells compared to WT-B8 cells. However, this difference in the rate of SSA repair is abrogated upon Recql5 knockdown, suggesting that Recql5 functions as a mediator for the SSA DNA repair pathway. Cumulatively, these findings suggest that Recql5 up-regulation in Jak2V617F-expressing cells plays a role in protecting against DNA damage-induced cell death through (1) stabilization of stalled replication forks thus preventing their collapse, and (2) promoting rapid (albeit error prone) SSA DNA repair to ameliorate genomic instability. Finally, we tested whether modulation of RECQL5 could also increase sensitivity of JAK2V617F-positive cells from primary MPN patients to HU. Following depletion with RECQL5, c-kit-enriched peripheral blood mononuclear cells from 2 essential thrombocythemia and 3 myelofibrosis patients were grown in semi-solid medium supplemented with HU for 14 days. Strikingly, we observed more specific eradication of JAK2V617F-positive erythroid progenitor colonies compared to autologous wildtype colonies from all patients examined. In contrast, no specific killing of JAK2V617F-positive erythroblasts was seen following transduction of control hairpins. This suggests that RECQL5 knockdown may potentially open a therapeutic window by sensitizing Jak2V617F-expressing cells to HU and other agents that induce replication stress. Disclosures No relevant conflicts of interest to declare.

scholarly journals The RECQL helicase prevents replication fork collapse during replication stress

2020 ◽  
Vol 3 (10) ◽  
pp. e202000668
Author(s):  
Bente Benedict ◽  
Marit AE van Bueren ◽  
Frank PA van Gemert ◽  
Cor Lieftink ◽  
Sergi Guerrero Llobet ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Replication Fork ◽  
Critical Role ◽  
Replication Stress ◽  
Strand Break ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Dna Dsbs ◽  
Fork Stalling ◽  
Stalled Replication Forks

Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy.

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