scholarly journals Destabilization of the holo-DNA Polδ by loss of Pol32 confers conditional lethality that can be suppressed by stabilizing Pol31-Pol3 interaction

2021 ◽  
Author(s):  
Kenji Shimada ◽  
Monika Tsai-Pflugfelder ◽  
Niloofar Davoodi Vijeh Motlagh ◽  
Neda Delgoshaie ◽  
Jeannette Fuchs ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Replication Fork ◽  
Essential Role ◽  
Genome Integrity ◽  
Genome Replication ◽  
Replication Forks ◽  
Kinase Activation ◽  
Yeast Strains ◽  
Stalled Replication Forks ◽  
Conserved Amino Acids

AbstractDNA Polymerase δ plays an essential role in genome replication and in the preservation of genome integrity. In S. cerevisiae, Polδ consists of three subunits: Pol3 (the catalytic subunit), Pol31 and Pol32. We have constructed pol31 mutants by alanine substitution at conserved amino acids, and identified three alleles that do not confer any disadvantage on their own, but which suppress the cold-, HU- and MMS-hypersensitivity of yeast strains lacking Pol32. We have shown that Pol31 and Pol32 are both involved in translesion synthesis, error-free bypass synthesis, and in preservation of replication fork stability under conditions of HU arrest. We identified a solvent exposed loop in Pol31 defined by two alanine substitutions at T415 and W417. Whereas pol31-T4l5A compromises polymerase stability at stalled forks, pol31-W417A is able to suppress many, but not all, of the phenotypes arising from pol32Δ. ChIP analyses showed that the absence of Pol32 destabilizes Pole and Polα at stalled replication forks, but does not interfere with checkpoint kinase activation. We show that the Pol31-W417A-mediated suppression of replicationstress sensitivity in pol32Δ stems from enhanced interaction between Pol3 and Pol31, which stabilizes a functional Polδ.

scholarly journals Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

2003 ◽  
Vol 185 (2) ◽  
pp. 630-644 ◽  
Author(s):  
Aline V. Grigorian ◽  
Rachel B. Lustig ◽  
Elena C. Guzmán ◽  
Joseph M. Mahaffy ◽  
Judith W. Zyskind
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Recombinational Repair ◽  
Replication Forks ◽  
Dna Replication Initiation ◽  
Escherichia Coli Cells ◽  
Repair Pathways ◽  
Stalled Replication Forks

ABSTRACT The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

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 Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

2019 ◽  
Author(s):  
Xinxing Lyu ◽  
Kai-Hang Lei ◽  
Olga Shiva ◽  
Megan Chastain ◽  
Peter Chi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Replication Fork ◽  
Genome Instability ◽  
Genome Integrity ◽  
Replication Forks ◽  
Cellular Sensitivity ◽  
Fork Stalling ◽  
Stalled Replication Forks ◽  
Nuclease Degradation

AbstractDegradation and collapse of stalled replication forks are main sources of genome instability, yet the molecular mechanism for protecting forks from degradation/collapse is not well understood. Here, we report that human CST (CTC1-STN1-TEN1), a single-stranded DNA binding protein complex, localizes at stalled forks and protects forks from MRE11 nuclease degradation upon replication perturbation. CST deficiency causes nascent strand degradation, ssDNA accumulation after fork stalling, and delay in replication recovery, leading to cellular sensitivity to fork stalling agents. Purified CST binds to 5’ overhangs and directly blocks MRE11 degradation in vitro, and the DNA binding ability of CST is required for blocking MRE11-mediated nascent strand degradation. Finally, we uncover that CST and BRCA2 form non-overlapping foci upon fork stalling, and CST inactivation is synthetic with BRCA2 deficiency in inducing genome instability. Collectively, our findings identify CST as an important fork protector to preserve genome integrity under replication perturbation.

scholarly journals Replication Fork Reversal and Protection

2021 ◽  
Vol 9 ◽  
Author(s):  
Shan Qiu ◽  
Guixing Jiang ◽  
Liping Cao ◽  
Jun Huang
Keyword(s):  
Mammalian Cells ◽  
Replication Fork ◽  
Protective Mechanism ◽  
Genome Replication ◽  
Replication Forks ◽  
Key Factors ◽  
Damage Repair ◽  
Brca1 Brca2 ◽  
Stalled Replication Forks ◽  
Nuclease Degradation

During genome replication, replication forks often encounter obstacles that impede their progression. Arrested forks are unstable structures that can give rise to collapse and rearrange if they are not properly processed and restarted. Replication fork reversal is a critical protective mechanism in higher eukaryotic cells in response to replication stress, in which forks reverse their direction to form a Holliday junction-like structure. The reversed replication forks are protected from nuclease degradation by DNA damage repair proteins, such as BRCA1, BRCA2, and RAD51. Some of these molecules work cooperatively, while others have unique functions. Once the stress is resolved, the replication forks can restart with the help of enzymes, including human RECQ1 helicase, but restart will not be considered here. Here, we review research on the key factors and mechanisms required for the remodeling and protection of stalled replication forks in mammalian cells.

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 Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks

2009 ◽  
Vol 23 (20) ◽  
pp. 2405-2414 ◽  
Author(s):  
C. E. Bansbach ◽  
R. Betous ◽  
C. A. Lovejoy ◽  
G. G. Glick ◽  
D. Cortez
Keyword(s):  
Genome Integrity ◽  
Replication Forks ◽  
Stalled Replication Forks

scholarly journals TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

2009 ◽  
Vol 184 (6) ◽  
pp. 793-804 ◽  
Author(s):  
Shan Yan ◽  
W. Matthew Michael
Keyword(s):  
Dna Polymerase ◽  
Replication Stress ◽  
Replication Forks ◽  
Ataxia Telangiectasia Mutated ◽  
Interacting Protein ◽  
Checkpoint Signaling ◽  
Binding Partner ◽  
Assembly Pathway ◽  
Egg Extracts ◽  
Stalled Replication Forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.

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