scholarly journals Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery

2019 ◽  
Vol 29 (7) ◽  
pp. 1083-1095 ◽  
Author(s):  
Maya Raghunandan ◽  
Jung Eun Yeo ◽  
Ryan Walter ◽  
Kai Saito ◽  
Adam J Harvey ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Replication Fork ◽  
Genome Stability ◽  
Chromosome Instability ◽  
Histone H3 ◽  
Underlying Mechanism ◽  
Replication Forks ◽  
Death Domain ◽  
Origin Firing ◽  
Stalled Replication Forks

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.

scholarly journals Checkpoint regulation of replication forks: global or local?

2013 ◽  
Vol 41 (6) ◽  
pp. 1701-1705 ◽  
Author(s):  
Divya Ramalingam Iyer ◽  
Nicholas Rhind
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Dna Damage Checkpoint ◽  
Replication Forks ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Late Origin ◽  
Stalled Replication Forks

Cell-cycle checkpoints are generally global in nature: one unattached kinetochore prevents the segregation of all chromosomes; stalled replication forks inhibit late origin firing throughout the genome. A potential exception to this rule is the regulation of replication fork progression by the S-phase DNA damage checkpoint. In this case, it is possible that the checkpoint is global, and it slows all replication forks in the genome. However, it is also possible that the checkpoint acts locally at sites of DNA damage, and only slows those forks that encounter DNA damage. Whether the checkpoint regulates forks globally or locally has important mechanistic implications for how replication forks deal with damaged DNA during S-phase.

scholarly journals SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

2020 ◽  
Author(s):  
Julie Rageul ◽  
Jennifer J. Park ◽  
Ping Ping Zeng ◽  
Eun-A Lee ◽  
Jihyeon Yang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Essential Role ◽  
Replication Stress ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Fork Protection Complex ◽  
Stalled Replication Forks ◽  
Stalled Fork

ABSTRACTProtecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

scholarly journals Emerging functions of Fanconi anemia genes in replication fork protection pathways

2020 ◽  
Vol 29 (R2) ◽  
pp. R158-R164 ◽  
Author(s):  
Arun Mouli Kolinjivadi ◽  
Wayne Crismani ◽  
Joanne Ngeow
Keyword(s):  
Dna Replication ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Genome Stability ◽  
Chromosome Instability ◽  
Replication Stress ◽  
Short Review ◽  
Gene Products ◽  
Dna Double Strand Breaks ◽  
Variants Of Unknown Significance

Abstract Germline mutations in Fanconi anemia (FA) genes predispose to chromosome instability syndromes, such as FA and cancers. FA gene products have traditionally been studied for their role in interstrand cross link (ICL) repair. A fraction of FA gene products are classical homologous recombination (HR) factors that are involved in repairing DNA double-strand breaks (DSBs) in an error-free manner. Emerging evidence suggests that, independent of ICL and HR repair, FA genes protect DNA replication forks in the presence of replication stress. Therefore, understanding the precise function of FA genes and their role in promoting genome stability in response to DNA replication stress is crucial for diagnosing FA and FA-associated cancers. Moreover, molecular understanding of the FA pathway will greatly help to establish proper functional assays for variants of unknown significance (VUS), often encountered in clinics. In this short review, we discuss the recently uncovered molecular details of FA genes in replication fork protection pathways. Finally, we examine how novel FA variants predispose to FA and cancer, due to defective replication fork protection activity.

scholarly journals SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Julie Rageul ◽  
Jennifer J. Park ◽  
Ping Ping Zeng ◽  
Eun-A Lee ◽  
Jihyeon Yang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Essential Role ◽  
Replication Stress ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Fork Protection Complex ◽  
Stalled Replication Forks ◽  
Stalled Fork

Abstract Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

scholarly journals MRNIP is a replication fork protection factor

2020 ◽  
Vol 6 (28) ◽  
pp. eaba5974 ◽  
Author(s):  
L. G. Bennett ◽  
A. M. Wilkie ◽  
E. Antonopoulou ◽  
I. Ceppi ◽  
A. Sanchez ◽  
...  
Keyword(s):  
Chromosomal Instability ◽  
Replication Fork ◽  
Chromosome Instability ◽  
Genome Integrity ◽  
Replication Forks ◽  
Protection Factor ◽  
Replication Fork Progression ◽  
Dna Junctions ◽  
Genomic Regions ◽  
Stalled Replication Forks

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease–dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.

scholarly journals Actin nucleation safeguards single-stranded DNA and promotes replication fork protection

2021 ◽  
Author(s):  
Jadwiga Nieminuszczy ◽  
Peter Martin ◽  
Ronan Broderick ◽  
Joanna Krwawicz ◽  
Alexandra Kanellou ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Stability ◽  
Actin Polymerization ◽  
Protein A ◽  
Genome Replication ◽  
Replication Forks ◽  
Actin Nucleation ◽  
Replicative Stress ◽  
Stalled Replication Forks ◽  
Polymeric Actin

Abstract Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress and protect stalled replication forks. All such responses rely on the formation of Replication Protein A (RPA)-ssDNA complexes, yet supra-physiological binding of RPA to ssDNA is toxic. How cells regulate RPA availability to promote fork protection and genome stability is largely unknown. Here we establish that during replication excess RPA is sequestered by monomeric actin and released upon replicative stress through transition to polymeric actin state. Impairment in actin nucleation leads to RPA sequestration, deprotection of ssDNA generated at the stressed forks and consequently, catastrophic fork collapse and hypersensitivity to replication inhibitors. In line with this, we show that increasing RPA load is sufficient to restore efficient fork protection in actin polymerization mutants. Collectively, this work identifies a simple yet robust RPA-buffering mechanism regulating its availability to bind ssDNA and protect replication forks against nucleolytic degradation. Inhibition of this pathway could be of therapeutic interest in treatment of cancers.

scholarly journals Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Christophe de La Roche Saint-André ◽  
Vincent Géli
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
Genetic Material ◽  
S Phase ◽  
Replication Forks ◽  
H3k4 Methylation ◽  
Origin Firing ◽  
Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

scholarly journals Acetylation modulates the Fanconi anemia pathway by protecting FAAP20 from ubiquitin-mediated proteasomal degradation

2020 ◽  
Vol 295 (40) ◽  
pp. 13887-13901
Author(s):  
Bhavika Nagareddy ◽  
Arafat Khan ◽  
Hyungjin Kim
Keyword(s):  
Fanconi Anemia ◽  
Posttranslational Modifications ◽  
Genome Stability ◽  
Chromosome Instability ◽  
Lysine Residue ◽  
Core Complex ◽  
Ubiquitin E3 Ligase ◽  
Pathway Activation ◽  
Anemia Group ◽  
Inherited Mutations

Fanconi anemia (FA) is a chromosome instability syndrome of children caused by inherited mutations in one of FA genes, which together constitute a DNA interstrand cross-link (ICL) repair, or the FA pathway. Monoubiquitination of Fanconi anemia group D2 protein (FANCD2) by the multisubunit ubiquitin E3 ligase, the FA core complex, is an obligate step in activation of the FA pathway, and its activity needs to be tightly regulated. FAAP20 is a key structural component of the FA core complex, and regulated proteolysis of FAAP20 mediated by prolyl cis-trans isomerization and phosphorylation at a consensus phosphodegron motif is essential for preserving the integrity of the FA core complex, and thus FANCD2 monoubiquitination. However, how ubiquitin-dependent FAAP20 degradation is modulated to fine-tune FA pathway activation remains largely un-known. Here, we present evidence that FAAP20 is acetylated by the acetyltransferase p300/CBP on lysine 152, the key residue that when polyubiquitinated results in the degradation of FAAP20. Acetylation or mutation of the lysine residue stabilizes FAAP20 by preventing its ubiquitination, thereby protecting it from proteasome-dependent FAAP20 degradation. Consequently, disruption of the FAAP20 acetylation pathway impairs FANCD2 activation. Together, our study reveals a competition mechanism between ubiquitination and acetylation of a common lysine residue that controls FAAP20 stability and highlights a complex balancing between different posttranslational modifications as a way to refine the FA pathway signaling required for DNA ICL repair and genome stability.

scholarly journals Exo1 phosphorylation inhibits exonuclease activity and prevents fork collapse in rad53 mutants independently of the 14-3-3 proteins

2020 ◽  
Vol 48 (6) ◽  
pp. 3053-3070
Author(s):  
Esther C Morafraile ◽  
Alberto Bugallo ◽  
Raquel Carreira ◽  
María Fernández ◽  
Cristina Martín-Castellanos ◽  
...  
Keyword(s):  
Genome Stability ◽  
Nuclease Activity ◽  
S Phase ◽  
Exonuclease Activity ◽  
Replication Forks ◽  
Post Translational Modifications ◽  
Replicative Stress ◽  
Stalled Replication Forks ◽  
Maintain Genome Stability ◽  
Specific Contribution

Abstract The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1–2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.

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