scholarly journals Roles of Claspin in regulation of DNA replication, replication stress responses and oncogenesis in human cells

2021 ◽  
Author(s):  
Hao-Wen Hsiao ◽  
Chi-Chun Yang ◽  
Hisao Masai
Keyword(s):  
Dna Replication ◽  
Stress Responses ◽  
Replication Fork ◽  
Therapeutic Potential ◽  
Genome Instability ◽  
Intramolecular Interaction ◽  
Replication Stress ◽  
Human Cells ◽  
Entire Genome ◽  
Replication Checkpoint

AbstractHuman cells need to cope with the stalling of DNA replication to complete replication of the entire genome to minimize genome instability. They respond to “replication stress” by activating the conserved ATR-Claspin-Chk1 replication checkpoint pathway. The stalled replication fork is detected and stabilized by the checkpoint proteins to prevent disintegration of the replication fork, to remove the lesion or problems that are causing fork block, and to facilitate the continuation of fork progression. Claspin, a factor conserved from yeasts to human, plays a crucial role as a mediator that transmits the replication fork arrest signal from the sensor kinase, ataxia telangiectasia and Rad3-related (ATR), to the effector kinase, Checkpoint kinase 1 (Chk1). Claspin interacts with multiple kinases and replication factors and facilitates efficient replication fork progression and initiation during the normal course of DNA replication as well. It interacts with Cdc7 kinase through the acidic patch segment near the C-terminus and this interaction is critical for efficient phosphorylation of Mcm in non-cancer cells and also for checkpoint activation. Phosphorylation of Claspin by Cdc7, recruited to the acidic patch, regulates the conformation of Claspin through affecting the intramolecular interaction between the N- and C-terminal segments of Claspin. Abundance of Claspin is regulated at both mRNA and protein levels (post-transcriptional regulation and protein stability) and affects the extent of replication checkpoint. In this article, we will discuss how the ATR-Claspin-Chk1 regulates normal and stressed DNA replication and provide insight into the therapeutic potential of targeting replication checkpoint for efficient cancer cell death.

scholarly journals MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

2018 ◽  
Author(s):  
Emily Yun-chia Chang ◽  
James P. Wells ◽  
Shu-Huei Tsai ◽  
Yan Coulombe ◽  
Yujia A. Chan ◽  
...  
Keyword(s):  
Dna Replication ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Genome Instability ◽  
Human Cancer ◽  
Replication Stress ◽  
Maintenance Factors ◽  
Genome Wide ◽  
A Genome ◽  
Dna Replication Stress

SUMMARYEctopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors such as RAD50. We show in yeast and human cells that R-loops accumulate during RAD50 depletion. In human cancer cell models, we find that RAD50 and its partners in the MRE11-RAD50-NBS1 complex regulate R-loop-associated DNA damage and replication stress. We show that a non-nucleolytic function of MRE11 is important for R-loop suppression via activation of PCNA-ubiquitination by RAD18 and recruiting anti-R-loop helicases in the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms of transcription-replication conflicts.

scholarly journals Cdc48 targets INQ-localized Mrc1 to facilitate recovery from replication stress

2021 ◽  
Author(s):  
Camilla Colding ◽  
Jacob Autzen ◽  
Boris Pfander ◽  
Michael Lisby
Keyword(s):  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
Methyl Methanesulfonate ◽  
Replication Checkpoint ◽  
Replication Restart ◽  
Efficient Replication ◽  
Dna Replication Stress ◽  
Stress Mediator ◽  
Control Compartment

DNA replication stress is a source of genome instability and a replication checkpoint has evolved to enable fork stabilisation and completion of replication during stress. Mediator of the replication checkpoint 1 (Mrc1) is the primary mediator of this response in Saccharomyces cerevisiae. Mrc1 is partially sequestered in the intranuclear quality control compartment (INQ) upon methyl methanesulfonate (MMS)-induced replication stress. Here we show that Mrc1 re-localizes from the replication fork to INQ during replication stress. Sequestration of Mrc1 in INQ is facilitated by the Btn2 chaperone and the Cdc48 segregase is required to release Mrc1 from INQ during recovery from replication stress. Consistently, we show that Cdc48 colocalizes with Mrc1 in INQ and we find that Mrc1 is recognized by the Cdc48 cofactors Ufd1 and Otu1, which contribute to clearance of Mrc1 from INQ. Our findings suggest that INQ localization of Mrc1 and Cdc48 function to facilitate replication stress recovery by transiently sequestering the replication checkpoint mediator Mrc1 and explains our observation that Btn2 and Cdc48 are required for efficient replication restart following MMS-induced replication stress.

scholarly journals Managing Single-Stranded DNA during Replication Stress in Fission Yeast

2021 ◽  
Author(s):  
Sarah A. Sabatinos ◽  
Susan L. Forsburg
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Single Stranded Dna ◽  
Replication Checkpoint ◽  
Primary Signal ◽  
Fork Stalling ◽  
Chromosome Integrity

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

scholarly journals Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae

2006 ◽  
Vol 175 (5) ◽  
pp. 729-741 ◽  
Author(s):  
Jorrit M. Enserink ◽  
Marcus B. Smolka ◽  
Huilin Zhou ◽  
Richard D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Morphology ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Checkpoint Proteins ◽  
Checkpoint Kinases ◽  
Dna Replication Checkpoint ◽  
Dna Replication Stress

In response to DNA replication stress in Saccharomyces cerevisiae, the DNA replication checkpoint maintains replication fork stability, prevents precocious chromosome segregation, and causes cells to arrest as large-budded cells. The checkpoint kinases Mec1 and Rad53 act in this checkpoint. Treatment of mec1 or rad53Δ mutants with replication inhibitors results in replication fork collapse and inappropriate partitioning of partially replicated chromosomes, leading to cell death. We describe a previously unappreciated function of various replication stress checkpoint proteins, including Rad53, in the control of cell morphology. Checkpoint mutants have aberrant cell morphology and cell walls, and show defective bud site selection. Rad53 shows genetic interactions with septin ring pathway components, and, along with other checkpoint proteins, controls the timely degradation of Swe1 during replication stress, thereby facilitating proper bud growth. Thus, checkpoint proteins play an important role in coordinating morphogenetic events with DNA replication during replication stress.

scholarly journals Managing Single-Stranded DNA during Replication Stress in Fission Yeast

2021 ◽  
Author(s):  
Sarah A. Sabatinos ◽  
Susan L. Forsburg
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Single Stranded Dna ◽  
Replication Checkpoint ◽  
Primary Signal ◽  
Fork Stalling ◽  
Chromosome Integrity

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

scholarly journals Active Replication Checkpoint Drives Genome Instability in Fission Yeast mcm4 Mutant

2020 ◽  
Vol 40 (14) ◽  
Author(s):  
Seong Min Kim ◽  
Susan L. Forsburg
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Genome Instability ◽  
Protein A ◽  
Replication Stress ◽  
Temperature Sensitive ◽  
Replication Checkpoint ◽  
Cycle Arrest ◽  
A Subunit ◽  
Replication Fork Arrest

ABSTRACT Upon replication fork arrest, the replication checkpoint kinase Cds1 is stimulated to preserve genome integrity. Robust activation of Cds1 in response to hydroxyurea prevents the endonuclease Mus81 from cleaving the stalled replication fork inappropriately. However, we find that the response is different in temperature-sensitive mcm4 mutants, affecting a subunit of the MCM replicative helicase. We show that Cds1 inhibition of Mus81 promotes genomic instability and allows mcm4-dg cells to evade cell cycle arrest. Cds1 regulation of Mus81 activity also contributes to the formation of the replication stress-induced DNA damage markers replication protein A (RPA) and Ku. These results identify a surprising role for Cds1 in driving DNA damage and disrupted chromosomal segregation under certain conditions of replication stress.

scholarly journals Detours to Replication: Functions of Specialized DNA Polymerases during Oncogene-induced Replication Stress

2018 ◽  
Vol 19 (10) ◽  
pp. 3255 ◽  
Author(s):  
Wei-Chung Tsao ◽  
Kristin Eckert
Keyword(s):  
Dna Replication ◽  
Stress Responses ◽  
Dna Polymerases ◽  
Repetitive Sequences ◽  
Replication Stress ◽  
Fragile Sites ◽  
Cancer Development ◽  
Cycle Arrest ◽  
Chromosomal Fragile Sites ◽  
Dna Secondary Structures

Incomplete and low-fidelity genome duplication contribute to genomic instability and cancer development. Difficult-to-Replicate Sequences, or DiToRS, are natural impediments in the genome that require specialized DNA polymerases and repair pathways to complete and maintain faithful DNA synthesis. DiToRS include non B-DNA secondary structures formed by repetitive sequences, for example within chromosomal fragile sites and telomeres, which inhibit DNA replication under endogenous stress conditions. Oncogene activation alters DNA replication dynamics and creates oncogenic replication stress, resulting in persistent activation of the DNA damage and replication stress responses, cell cycle arrest, and cell death. The response to oncogenic replication stress is highly complex and must be tightly regulated to prevent mutations and tumorigenesis. In this review, we summarize types of known DiToRS and the experimental evidence supporting replication inhibition, with a focus on the specialized DNA polymerases utilized to cope with these obstacles. In addition, we discuss different causes of oncogenic replication stress and its impact on DiToRS stability. We highlight recent findings regarding the regulation of DNA polymerases during oncogenic replication stress and the implications for cancer development.

scholarly journals A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression

2014 ◽  
Vol 42 (9) ◽  
pp. 5605-5615 ◽  
Author(s):  
Spencer W. Luebben ◽  
Tsuyoshi Kawabata ◽  
Charles S. Johnson ◽  
M. Gerard O'Sullivan ◽  
Naoko Shima
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Fork Progression

scholarly journals Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
André Franz ◽  
Paul A. Pirson ◽  
Domenic Pilger ◽  
Swagata Halder ◽  
Divya Achuthankutty ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dynamic Process ◽  
Metabolic Pathways ◽  
Replication Fork ◽  
Genome Instability ◽  
Genome Integrity ◽  
Substrate Selection ◽  
Replication Fork Progression ◽  
Replication Factors ◽  
Spatiotemporal Control

Abstract The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.

scholarly journals Transcriptome Analysis of Escherichia coli during dGTP Starvation

2016 ◽  
Vol 198 (11) ◽  
pp. 1631-1644 ◽  
Author(s):  
Mark Itsko ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Stress Responses ◽  
De Novo ◽  
Replication Stress ◽  
Building Blocks ◽  
Content Type ◽  
Final Cause ◽  
E Coli ◽  
Genome Wide

ABSTRACTOur laboratory recently discovered thatEscherichia colicells starved for the DNA precursor dGTP are killed efficiently (dGTP starvation) in a manner similar to that described for thymineless death (TLD). Conditions for specific dGTP starvation can be achieved by depriving anE. colioptA1 gptstrain of the purine nucleotide precursor hypoxanthine (Hx). To gain insight into the mechanisms underlying dGTP starvation, we conducted genome-wide gene expression analyses of actively growingoptA1 gptcells subjected to hypoxanthine deprivation for increasing periods. The data show that upon Hx withdrawal, theoptA1 gptstrain displays a diminished ability to derepress thede novopurine biosynthesis genes, likely due to internal guanine accumulation. The impairment in fully inducing thepurRregulon may be a contributing factor to the lethality of dGTP starvation. At later time points, and coinciding with cell lethality, strong induction of the SOS response was observed, supporting the concept of replication stress as a final cause of death. No evidence was observed in the starved cells for the participation of other stress responses, including therpoS-mediated global stress response, reinforcing the lack of feedback of replication stress to the global metabolism of the cell. The genome-wide expression data also provide direct evidence for increased genome complexity during dGTP starvation, as a markedly increased gradient was observed for expression of genes located near the replication origin relative to those located toward the replication terminus.IMPORTANCEControl of the supply of the building blocks (deoxynucleoside triphosphates [dNTPs]) for DNA replication is important for ensuring genome integrity and cell viability. When cells are starved specifically for one of the four dNTPs, dGTP, the process of DNA replication is disturbed in a manner that can lead to eventual death. In the present study, we investigated the transcriptional changes in the bacteriumE. coliduring dGTP starvation. The results show increasing DNA replication stress with an increased time of starvation, as evidenced by induction of the bacterial SOS system, as well as a notable lack of induction of other stress responses that could have saved the cells from cell death by slowing down cell growth.

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