FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress

Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)–binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress.

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The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

mBio ◽

10.1128/mbio.01192-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 5

Author(s):

Alex Bronstein ◽

Lihi Gershon ◽

Gilad Grinberg ◽

Elisa Alonso-Perez ◽

Martin Kupiec

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Genome Stability ◽

Dna Helicase ◽

Main Role ◽

Helicase Domain ◽

Helicase Activity ◽

C Terminus ◽

Srs2 Helicase

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. TheSaccharomyces cerevisiaeyeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a newSRS2allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of thesrs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.IMPORTANCEHomologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

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ARID1A regulates R-loop associated DNA replication stress

10.1101/2020.11.16.384446 ◽

2020 ◽

Author(s):

Shuhe Tsai ◽

Emily Yun-chia Chang ◽

Louis-Alexandre Fournier ◽

James P. Wells ◽

Sean W. Minaker ◽

...

Keyword(s):

Dna Replication ◽

Genome Stability ◽

Clear Cell ◽

Replication Stress ◽

Clear Cell Ovarian Carcinoma ◽

Dna Replication Stress ◽

Cancer Types ◽

The Impact ◽

Oncogenic Gene ◽

Transcription And Replication

ABSTRACTARID1A is lost in up to 7% of all cancers, and this frequency increases in certain cancer types, such as clear cell ovarian carcinoma where ARID1A protein is lost in about 50% of cases. While the impact of ARID1A loss on the function of the BAF chromatin remodeller complexes is likely to drive oncogenic gene expression programs in specific contexts, ARID1A also binds genome stability regulators such as ATR and TOP2. Here we show that ARID1A loss leads to DNA replication stress associated with R-loops and transcription-replication conflicts in human cells. These effects correlate with altered transcription and replication dynamics in ARID1A knockout cells and to reduced TOP2A binding at R-loop sites. Together this work extends mechanisms of replication stress in ARID1A deficient cells with implications for targeting ARID1A deficient cancers.

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Histone dynamics during DNA replication stress

Journal of Biomedical Science ◽

10.1186/s12929-021-00743-5 ◽

2021 ◽

Vol 28 (1) ◽

Author(s):

Chia-Ling Hsu ◽

Shin Yen Chong ◽

Chia-Yeh Lin ◽

Cheng-Fu Kao

Keyword(s):

Stress Responses ◽

Genome Stability ◽

Genome Instability ◽

Replication Stress ◽

Dna Lesions ◽

Protective Mechanisms ◽

Replication Process ◽

Dna Replication Stress ◽

External Sources

AbstractAccurate and complete replication of the genome is essential not only for genome stability but also for cell viability. However, cells face constant threats to the replication process, such as spontaneous DNA modifications and DNA lesions from endogenous and external sources. Any obstacle that slows down replication forks or perturbs replication dynamics is generally considered to be a form of replication stress, and the past decade has seen numerous advances in our understanding of how cells respond to and resolve such challenges. Furthermore, recent studies have also uncovered links between defects in replication stress responses and genome instability or various diseases, such as cancer. Because replication stress takes place in the context of chromatin, histone dynamics play key roles in modulating fork progression and replication stress responses. Here, we summarize the current understanding of histone dynamics in replication stress, highlighting recent advances in the characterization of fork-protective mechanisms.

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Global analysis of genomic instability caused by DNA replication stress in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618129113 ◽

2016 ◽

Vol 113 (50) ◽

pp. E8114-E8121 ◽

Cited By ~ 28

Author(s):

Dao-Qiong Zheng ◽

Ke Zhang ◽

Xue-Chang Wu ◽

Piotr A. Mieczkowski ◽

Thomas D. Petes

Keyword(s):

Dna Replication ◽

Genomic Instability ◽

Dna Polymerase ◽

Replication Stress ◽

Dna Lesions ◽

Chromosome Loss ◽

Dna Breaks ◽

Dna Polymerase Δ ◽

Dna Replication Stress ◽

Low Levels

DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G2 phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.

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Rfa2 is specifically dephosphorylated by Pph3 in Candida albicans

Biochemical Journal ◽

10.1042/bj20120952 ◽

2013 ◽

Vol 449 (3) ◽

pp. 673-681 ◽

Cited By ~ 9

Author(s):

Haitao Wang ◽

Jiaxin Gao ◽

Ada Hang-Heng Wong ◽

Kangdi Hu ◽

Wanjie Li ◽

...

Keyword(s):

Candida Albicans ◽

Dna Replication ◽

Protein A ◽

Replication Stress ◽

Dna Interaction ◽

G1 Phase ◽

Protein Protein Interaction ◽

Double Stranded Dna ◽

Dna Replication Stress ◽

G1 Cells

Rfa2 is a ssDNA (single-stranded DNA)-binding protein that plays an important role in DNA replication, recombination and repair. Rfa2 is regulated by phosphorylation, which alters its protein–protein interaction and protein–DNA interaction. In the present study, we found that the Pph3–Psy2 phosphatase complex is responsible for Rfa2 dephosphorylation both during normal G1-phase and under DNA replication stress in Candida albicans. Phosphorylated Rfa2 extracted from pph3Δ or psy2Δ G1 cells exhibited diminished binding affinity to dsDNA (double-stranded DNA) but not to ssDNA. We also discovered that Cdc28 (cell division cycle 28) and Mec1 are responsible for Rfa2 phosphorylation in G1-phase and under DNA replication stress respectively. Moreover, MS revealed that the domain of Rfa2 that was phosphorylated in G1-phase differed from that phosphorylated under the stress conditions. The results of the present study imply that differential phosphorylation plays a crucial role in RPA (replication protein A) regulation.

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Interplay between DNA replication stress, chromatin dynamics and DNA-damage response for the maintenance of genome stability

Mutation Research/Reviews in Mutation Research ◽

10.1016/j.mrrev.2020.108346 ◽

2021 ◽

Vol 787 ◽

pp. 108346

Author(s):

Maddalena Mognato ◽

Susanne Burdak-Rothkamm ◽

Kai Rothkamm

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Genome Stability ◽

Replication Stress ◽

Chromatin Dynamics ◽

Damage Response ◽

Dna Replication Stress

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Characterization of Streptococcus pneumoniae PriA helicase and its ATPase and unwinding activities in DNA replication restart

Biochemical Journal ◽

10.1042/bcj20200269 ◽

2020 ◽

Vol 477 (19) ◽

pp. 3911-3922

Author(s):

Min-Guan Lin ◽

Yi-Ching Li ◽

Chwan-Deng Hsiao

Keyword(s):

Streptococcus Pneumoniae ◽

Dna Replication ◽

Dna Binding ◽

Protein A ◽

Gc Content ◽

Dna Helicase ◽

Dna Lesions ◽

Loop Area ◽

Replication Restart ◽

Key Factor

DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein — a 3′ to 5′ superfamily-2 DNA helicase — is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities of Streptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses. By comparing various DNA substrates, we observed that SpPriA is unable to unwind duplex DNA with high GC content. We constructed a deletion mutant protein (SpPriAdeloop) from which the loop area of the DNA-binding domain of PriA had been removed. Functional assays on SpPriAdeloop revealed that the loop area is important in endowing DNA-binding properties on the helicase. We also show that the presence of DnaD loader protein is important for enhancing SpPriA ATPase and DNA unwinding activities.

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LIM Protein Ajuba associates with the RPA complex through direct and cell cycle-dependent interaction with the RPA70 subunit

10.1101/177840 ◽

2017 ◽

Author(s):

Sandy Fowler ◽

Pascal Maguin ◽

Sampada Kalan ◽

Diego Loayza

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Genome Stability ◽

Replication Stress ◽

Cellular Transformation ◽

Early Event ◽

Lim Protein ◽

Show Evidence ◽

Dna Replication Stress ◽

Cycle Dependent

AbstractDNA damage response pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress. An early event in this activation is the recruitment and phosphorylation of RPA, a single stranded DNA binding complex composed of three subunits, RPA70,RPA32 and RPA14. We have previously shown that the LIM protein Ajuba associates with RPA, and that depletion of Ajuba leads to potent activation of ATR. In this study, we show evidence that the Ajuba-RPA interaction occurs through direct protein contact with RPA70, and that their association is cell cycle-regulated and is reduced upon DNA replication stress. We propose a model in which Ajuba negatively regulates the ATR pathway by directly interacting with RPA70, thereby preventing an inappropriate ATR activation. Our results provide a framework to understand the mechanism of regulation of ATR in human cells, which is important to prevent cellular transformation and tumorigenesis.

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Direct quantification of the translocation activities of Saccharomyces cerevisiae Pif1 helicase

Nucleic Acids Research ◽

10.1093/nar/gkz541 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7494-7501

Author(s):

Chen Lu ◽

Shimin Le ◽

Jin Chen ◽

Alicia K Byrd ◽

Daniela Rhodes ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Genome Stability ◽

Dna Helicase ◽

Telomere Maintenance ◽

The Other ◽

Biological Processes ◽

Helicase Activity ◽

Pif1 Helicase ◽

Direct Quantification

AbstractSaccharomyces cerevisiae Pif1 (ScPif1) is known as an ATP-dependent DNA helicase that plays critical roles in a number of important biological processes such as DNA replication, telomere maintenance and genome stability maintenance. Besides its DNA helicase activity, ScPif1 is also known as a single-stranded DNA (ssDNA) translocase, while how ScPif1 translocates on ssDNA is unclear. Here, by measuring the translocation activity of individual ScPif1 molecules on ssDNA extended by mechanical force, we identified two distinct types of ssDNA translocation. In one type, ScPif1 moves along the ssDNA track with a rate of ∼140 nt/s in 100 μM ATP, whereas in the other type, ScPif1 is immobilized to a fixed location of ssDNA and generates ssDNA loops against force. Between the two, the mobile translocation is the major form at nanomolar ScPif1 concentrations although patrolling becomes more frequent at micromolar concentrations. Together, our results suggest that ScPif1 translocates on extended ssDNA in two distinct modes, primarily in a ‘mobile’ manner.

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