scholarly journals DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress

2020 ◽  
Vol 117 (52) ◽  
pp. 33436-33445
Author(s):  
Rui Wang ◽  
Walter F. Lenoir ◽  
Chao Wang ◽  
Dan Su ◽  
Megan McLaughlin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Dna Polymerase ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Germ Line ◽  
Synthetic Lethality ◽  
Critical Role ◽  
Replication Stress ◽  
Loss Of Function

Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4. As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.

scholarly journals SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

2020 ◽  
Vol 220 (1) ◽  
Author(s):  
Riko Ishimoto ◽  
Yota Tsuzuki ◽  
Tomoki Matsumura ◽  
Seiichiro Kurashige ◽  
Kouki Enokitani ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Interactions ◽  
Chromosomal Instability ◽  
Replication Fork ◽  
Protein Complexes ◽  
Critical Role ◽  
Replication Stress ◽  
S Phase ◽  
Damage Response

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA–protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4–XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4–ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.

scholarly journals SAMHD1 promotes oncogene-induced replication stress

2020 ◽  
Author(s):  
Si Min Zhang ◽  
Jose M Calderón-Montaño ◽  
Sean G Rudd
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Overall Survival ◽  
Dna Replication ◽  
Replication Fork ◽  
Potential Role ◽  
Human Fibroblasts ◽  
Replication Stress ◽  
Oncogenic Ras ◽  
Dna Replication Stress

AbstractOncogenes induce DNA replication stress in cancer cells. Although this was established more than a decade ago, we are still unravelling the molecular underpinnings of this phenomenon, which will be critical if we are to exploit this knowledge to improve cancer treatment. A key mediator of oncogene-induced replication stress is the availability of DNA precursors, which will limit ongoing DNA synthesis by cellular replicases. In this study, we identify a potential role for nucleotide catabolism in promoting replication stress induced by oncogenes. Specifically, we establish that the dNTPase SAMHD1 slows DNA replication fork speeds in human fibroblasts harbouring an oncogenic RAS allele, elevating levels of endogenous DNA damage, and ultimately limiting cell proliferation. We then show that oncogenic RAS-driven tumours express reduced SAMHD1 levels, suggesting they have overcome this tumour suppressor barrier, and that this correlates with worse overall survival for these patients.Abstract Figure

AMPK blocks chromatin activation and consequent R-loop formation to protect genome stability following acute starvation

2021 ◽  
Author(s):  
Bing Sun ◽  
McLean Sherrin ◽  
Richard Roy
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Germ Line ◽  
Critical Role ◽  
Significant Proportion ◽  
Loop Formation ◽  
C Elegans ◽  
Chromatin Activation ◽  
Acute Starvation

Abstract During periods of starvation organisms must modify both gene expression and metabolic pathways to adjust to the energy stress. We previously reported that C. elegans that lack AMPK have transgenerational reproductive defects that result from abnormally elevated H3K4me3 levels in the germ line following recovery from acute starvation1. Here we show that H3K4me3 is dramatically increased at promoters, driving aberrant transcription elongation that results in the accumulation of R-loops in the starved AMPK mutants. DRIP-seq analysis demonstrated that a significant proportion of the genome was affected by R-loop formation with a dramatic expansion in the number of R-loops at numerous loci, most pronounced at the promoter-TSS regions of genes in the starved AMPK mutants. The R-loops are transmissible into subsequent generations, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK null germ lines show considerably more RAD-51 foci at sites of R-loop formation, potentially sequestering it from its critical role at meiotic breaks and/or at sites of induced DNA damage. Our study reveals a previously unforeseen role of AMPK in maintaining genome stability following starvation, where in its absence R-loops accumulate, resulting in reproductive compromise and DNA damage hypersensitivity.

scholarly journals DNA Repair: Exploiting the Fanconi Anemia Pathway As a Potential Therapeutic Target

2011 ◽  
pp. 453-465 ◽  
Author(s):  
T. HUCL ◽  
E. GALLMEIER
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Fanconi Anemia ◽  
Cancer Susceptibility ◽  
Synthetic Lethality ◽  
Bone Marrow Failure ◽  
Long Term Effects ◽  
Alternative Pathways ◽  
Repair Mechanisms ◽  
Relationship Of

DNA repair is an active cellular process to respond to constant DNA damage caused by metabolic processes and environmental factors. Since the outcome of DNA damage is generally adverse and long term effects may contribute to oncogenesis, cells have developed a variety of DNA repair mechanisms, which operate depending on the type of DNA damage inflicted. At least 15 Fanconi anemia (FA) proteins interact in a common pathway involved in homologous recombination. Inherited homozygous mutations in any of these FA genes cause a rare disease, Fanconi anemia, characterized by congenital abnormalities, progressive bone-marrow failure and cancer susceptibility. Heterozygous germline FA mutations predispose to various types of cancer. In addition, somatic FA mutations have been identified in diverse cancer types. Evidence exists that cells deficient in the FA pathway become dependent on alternative pathways for survival. Additional inhibition of such alternative pathways is thus expected to result in cell death, creating a relationship of synthetic lethality. Identifying these relationships can reveal yet unknown mechanisms of DNA repair and new targets for therapy.

scholarly journals Class VI Unconventional Myosin is Required for Spermatogenesis inDrosophila

1999 ◽  
Vol 10 (12) ◽  
pp. 4341-4353 ◽  
Author(s):  
Jennifer L. Hicks ◽  
Wu-Min Deng ◽  
Aaron D. Rogat ◽  
Kathryn G. Miller ◽  
Mary Bownes
Keyword(s):  
Male Sterility ◽  
Germ Line ◽  
Critical Role ◽  
Leading Edge ◽  
Cytoskeletal Proteins ◽  
Loss Of Function ◽  
Unconventional Myosin ◽  
Membrane Reorganization ◽  
Cytoplasmic Bridges ◽  
Mitosis And Meiosis

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

scholarly journals Holding All the Cards—How Fanconi Anemia Proteins Deal with Replication Stress and Preserve Genomic Stability

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 170 ◽  
Author(s):  
Arindam Datta ◽  
Robert M. Brosh Jr.
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Excision Repair ◽  
Double Helix ◽  
Bone Marrow Failure ◽  
Molecular Genetic ◽  
Replication Stress ◽  
Gene Products ◽  
Hematopoietic Stem ◽  
Dna Double Helix

Fanconi anemia (FA) is a hereditary chromosomal instability disorder often displaying congenital abnormalities and characterized by a predisposition to progressive bone marrow failure (BMF) and cancer. Over the last 25 years since the discovery of the first linkage of genetic mutations to FA, its molecular genetic landscape has expanded tremendously as it became apparent that FA is a disease characterized by a defect in a specific DNA repair pathway responsible for the correction of covalent cross-links between the two complementary strands of the DNA double helix. This pathway has become increasingly complex, with the discovery of now over 20 FA-linked genes implicated in interstrand cross-link (ICL) repair. Moreover, gene products known to be involved in double-strand break (DSB) repair, mismatch repair (MMR), and nucleotide excision repair (NER) play roles in the ICL response and repair of associated DNA damage. While ICL repair is predominantly coupled with DNA replication, it also can occur in non-replicating cells. DNA damage accumulation and hematopoietic stem cell failure are thought to contribute to the increased inflammation and oxidative stress prevalent in FA. Adding to its confounding nature, certain FA gene products are also engaged in the response to replication stress, caused endogenously or by agents other than ICL-inducing drugs. In this review, we discuss the mechanistic aspects of the FA pathway and the molecular defects leading to elevated replication stress believed to underlie the cellular phenotypes and clinical features of FA.

scholarly journals Supraphysiological protection from replication stress does not extend mammalian lifespan

2020 ◽  
Author(s):  
Eliene Albers ◽  
Alexandra Avram ◽  
Mauro Sbroggio ◽  
Oscar Fernandez-Capetillo ◽  
Andres J Lopez-Contreras
Keyword(s):  
Dna Damage ◽  
Transgenic Mice ◽  
Genomic Instability ◽  
Genetic Background ◽  
Replication Fork ◽  
Accelerated Aging ◽  
Replication Stress ◽  
Term Survival ◽  
Survival Studies

AbstractReplication Stress (RS) is a type of DNA damage generated at the replication fork, characterized by single-stranded DNA (ssDNA) accumulation, and which can be caused by a variety of factors. Previous studies have reported elevated RS levels in aged cells. In addition, mouse models with a deficient RS response show accelerated aging. However, the relevance of endogenous or physiological RS, compared to other sources of genomic instability, for the normal onset of aging is unknown. We have performed long term survival studies of transgenic mice with extra copies of the Chk1 and/or Rrm2 genes, which we previously showed extend the lifespan of a progeroid ATR-hypomorphic model suffering from high levels of RS. In contrast to their effect in the context of progeria, the lifespan of Chk1, Rrm2 and Chk1/Rrm2 transgenic mice was similar to WT littermates in physiological settings. Most mice studied died due to tumors -mainly lymphomas-irrespective of their genetic background. Interestingly, a slightly higher percentage of transgenic mice developed tumors compared to WT mice. Our results indicate that supraphysiological protection from RS does not extend lifespan, indicating that RS may not be a relevant source of genomic instability on the onset of “normal” aging.

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 CHK1 inhibition exacerbates replication stress induced by IGF blockade

Oncogene ◽  
2021 ◽  
Author(s):  
Xiaoning Wu ◽  
Elena Seraia ◽  
Stephanie B. Hatch ◽  
Xiao Wan ◽  
Daniel V. Ebner ◽  
...  
Keyword(s):  
Ribonucleotide Reductase ◽  
Replication Fork ◽  
Synthetic Lethality ◽  
Growth Factor Receptor ◽  
Replication Stress ◽  
Regulatory Subunit ◽  
Breast Cancer Cell Lines ◽  
Replication Fork Progression ◽  
Deoxynucleotide Triphosphate

AbstractWe recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

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