scholarly journals PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

2019 ◽  
Author(s):  
Tanay Thakar ◽  
Wendy Leung ◽  
Claudia M. Nicolae ◽  
Kristen E. Clements ◽  
Binghui Shen ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Normal Growth ◽  
Genotoxic Stress ◽  
Growth Conditions ◽  
Parp Inhibitors ◽  
Replication Forks ◽  
Lesion Bypass ◽  
Okazaki Fragment ◽  
Okazaki Fragment Maturation ◽  
Stalled Replication Forks

AbstractUpon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing ubiquitination-deficient 293T and RPE1 cells generated through CRISPR/Cas9 genome editing, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-mediated but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork, and incomplete Okazaki fragment synthesis and maturation, thus interfering with efficient PCNA unloading by ATAD5 and subsequent nucleosomal deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and BRCA2 results in a synergistic increase in nascent DNA degradation and sensitivity to PARP-inhibitors. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.

scholarly journals Phosphorylation of Rad55 on Serines 2, 8, and 14 Is Required for Efficient Homologous Recombination in the Recovery of Stalled Replication Forks

2006 ◽  
Vol 26 (22) ◽  
pp. 8396-8409 ◽  
Author(s):  
Kristina Herzberg ◽  
Vladimir I. Bashkirov ◽  
Michael Rolfsmeier ◽  
Edwin Haghnazari ◽  
W. Hayes McDonald ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Cellular Response ◽  
Replication Fork ◽  
Normal Growth ◽  
Genotoxic Stress ◽  
Replication Forks ◽  
Replication Fork Stalling ◽  
Fork Stalling ◽  
Stalled Replication Forks

ABSTRACT DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.

scholarly journals DisA limits RecA- and RadA/Sms-mediated replication fork remodelling to prevent genome instability

2020 ◽  
Author(s):  
Rubén Torres ◽  
Juan C. Alonso
Keyword(s):  
Genome Instability ◽  
Dna Helicase ◽  
Template Switching ◽  
Replication Forks ◽  
Lesion Bypass ◽  
Branched Dna ◽  
Subject Categories ◽  
Negative Effect ◽  
Dna Replication Stress ◽  
Stalled Replication Forks

AbstractThe DisA diadenylate cyclase (DAC), the DNA helicase RadA/Sms and the RecA recombinase are required to prevent a DNA replication stress during the revival of haploid Bacillus subtilis spores. Moreover, disA, radA and recA are epistatic among them in response to DNA damage. We show that DisA inhibits the ATPase activity of RadA/Sms C13A by competing for single-stranded (ss) DNA. In addition, DisA inhibits the helicase activity of RadA/Sms. RecA filamented onto ssDNA interacts with and recruits DisA and RadA/Sms onto branched DNA intermediates. In fact, RecA binds a reversed fork and facilitates RadA/Sms-mediated unwinding to restore a 3′-fork intermediate, but DisA inhibits it. Finally, RadA/Sms inhibits DisA DAC activity, but RecA counters this negative effect. We propose that RecA, DisA and RadA/Sms interactions, which are mutually exclusive, limit remodelling of stalled replication forks. DisA, in concert with RecA and/or RadA/Sms, indirectly contributes to template switching or lesion bypass, prevents fork breakage and facilitates the recovery of c-di-AMP levels to re-initiate cell proliferation.Subject CategoriesGenomic stability & Dynamics

scholarly journals Stxbp4 Regulates ΔNp63 Stability by Suppression of RACK1-Dependent Degradation

2009 ◽  
Vol 29 (14) ◽  
pp. 3953-3963 ◽  
Author(s):  
Yingchun Li ◽  
Melissa J. Peart ◽  
Carol Prives
Keyword(s):  
Squamous Cell Carcinoma ◽  
Normal Growth ◽  
Genotoxic Stress ◽  
Growth Conditions ◽  
Carcinoma Cells ◽  
Scaffold Proteins ◽  
Proliferating Cells ◽  
Protein Levels ◽  
Response To Stress

ABSTRACT p63, a member of the p53 tumor suppressor family, is essential for the development of epidermis as well as other stratified epithelia. Collective evidence indicates that ΔNp63 proteins, the N-terminally deleted versions of p63, are essential for the proliferation and survival of stratified epithelial cells and squamous cell carcinoma cells. But in response to DNA damage, ΔNp63 proteins are quickly downregulated in part through protein degradation. To elucidate the mechanisms by which ΔNp63 proteins are maintained at relatively high levels in proliferating cells but destabilized in response to stress, we sought to identify p63 interactive proteins that regulate p63 stability. We found that Stxbp4 and RACK1, two scaffold proteins, play central roles in balancing ΔNp63 protein levels. While Stxbp4 functions to stabilize ΔNp63 proteins, RACK1 targets ΔNp63 for degradation. Under normal growth conditions, Stxbp4 is indispensable for maintaining high basal levels of ΔNp63 and preventing RACK1-mediated p63 degradation. Upon genotoxic stress, however, Stxbp4 itself is downregulated, correlating with ΔNp63 destabilization mediated in part by RACK1. Taken together, we have delineated key mechanisms that regulate ΔNp63 protein stability in vivo.

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.

scholarly journals Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tanay Thakar ◽  
Wendy Leung ◽  
Claudia M. Nicolae ◽  
Kristen E. Clements ◽  
Binghui Shen ◽  
...  
Keyword(s):  
Chromatin Assembly ◽  
Replication Forks ◽  
Okazaki Fragment ◽  
Okazaki Fragment Maturation

scholarly journals Prevention of unwanted recombination at damaged replication forks

2020 ◽  
Vol 66 (6) ◽  
pp. 1045-1051 ◽  
Author(s):  
Carl P. Lehmann ◽  
Alberto Jiménez-Martín ◽  
Dana Branzei ◽  
José Antonio Tercero
Keyword(s):  
Dna Damage ◽  
Chromosome Replication ◽  
Dna Lesions ◽  
Template Switching ◽  
Replication Forks ◽  
Lesion Bypass ◽  
Alternative Mode ◽  
Replicative Stress ◽  
Dna Lesion ◽  
Stalled Replication Forks

Abstract Homologous recombination is essential for the maintenance of genome integrity but must be strictly controlled to avoid dangerous outcomes that produce the opposite effect, genomic instability. During unperturbed chromosome replication, recombination is globally inhibited at ongoing DNA replication forks, which helps to prevent deleterious genomic rearrangements. This inhibition is carried out by Srs2, a helicase that binds to SUMOylated PCNA and has an anti-recombinogenic function at replication forks. However, at damaged stalled forks, Srs2 is counteracted and DNA lesion bypass can be achieved by recombination-mediated template switching. In budding yeast, template switching is dependent on Rad5. In the absence of this protein, replication forks stall in the presence of DNA lesions and cells die. Recently, we showed that in cells lacking Rad5 that are exposed to DNA damage or replicative stress, elimination of the conserved Mgs1/WRNIP1 ATPase allows an alternative mode of DNA damage bypass that is driven by recombination and facilitates completion of chromosome replication and cell viability. We have proposed that Mgs1 is important to prevent a potentially harmful salvage pathway of recombination at damaged stalled forks. In this review, we summarize our current understanding of how unwanted recombination is prevented at damaged stalled replication forks.

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