Histone Ubiquitination by the DNA Damage Response Is Required for Efficient DNA Replication in Unperturbed S Phase

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

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Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525620113 ◽

2016 ◽

Vol 113 (26) ◽

pp. E3676-E3685 ◽

Cited By ~ 8

Author(s):

Nicholas A. Willis ◽

Chunshui Zhou ◽

Andrew E. H. Elia ◽

Johanne M. Murray ◽

Antony M. Carr ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Dna Damage Response ◽

Cellular Response ◽

Genome Stability ◽

Biological Significance ◽

S Phase ◽

Damage Response

The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase–specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes.

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Replication fork dynamics and the DNA damage response

Biochemical Journal ◽

10.1042/bj20112100 ◽

2012 ◽

Vol 443 (1) ◽

pp. 13-26 ◽

Cited By ~ 77

Author(s):

Rebecca M. Jones ◽

Eva Petermann

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Replication Fork ◽

Molecular Mechanisms ◽

S Phase ◽

Replication Initiation ◽

Developmental Defects ◽

Replication Fork Progression ◽

Damage Response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

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Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.00872-09 ◽

2010 ◽

Vol 30 (7) ◽

pp. 1607-1619 ◽

Cited By ~ 16

Author(s):

Cyril Ramathal ◽

Indrani C. Bagchi ◽

Milan K. Bagchi

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Epithelial Cells ◽

Cell Cycle Arrest ◽

Dna Damage Response ◽

S Phase ◽

Uterine Epithelial Cells ◽

Cycle Arrest ◽

Damage Response

ABSTRACT Female mice lacking the transcription factor C/EBPβ are infertile and display markedly reduced estrogen (E)-induced proliferation of the uterine epithelial lining during the reproductive cycle. The present study showed that E-stimulated luminal epithelial cells of a C/EBPβ-null uterus are able to proceed through the G1 phase of the cell cycle before getting arrested in the S phase. This cell cycle arrest was accompanied by markedly reduced levels of expression of E2F3, an E2F family member, and a lack of nuclear localization of cyclin E, a critical regulator of cdk2. An increased nuclear accumulation of p27, an inhibitor of the cyclin E-cdk2 complex, was also observed for the mutant epithelium. Gene expression profiling of C/EBPβ-null uterine epithelial cells revealed that the blockade of E-induced DNA replication triggers the activation of several well-known components of the DNA damage response pathway, such as ATM, ATR, histone H2AX, checkpoint kinase 1, and tumor suppressor p53. The activation of p53 by ATM/ATR kinase led to increased levels of expression of p21, an inhibitor of G1-S-phase progression, which helps maintain cell cycle arrest. Additionally, p53-dependent mechanisms contributed to an increased apoptosis of replication-defective cells in the C/EBPβ-null epithelium. C/EBPβ, therefore, is an essential mediator of E-induced growth and survival of uterine epithelial cells of cycling mice.

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Gene co-expression analysis applied to RNASEH2A reveals functional networks associated with DNA replication, DNA damage response, as well as cell cycle regulation

10.26226/morressier.5ebd45acffea6f735881b15e ◽

2020 ◽

Author(s):

Stefania Marsili

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Expression Analysis ◽

Cell Cycle Regulation ◽

Functional Networks ◽

Damage Response ◽

Cycle Regulation

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Cell cycle inertia underlies a bifurcation in cell fates after DNA damage

Science Advances ◽

10.1126/sciadv.abe3882 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabe3882

Author(s):

Jenny F. Nathans ◽

James A. Cornwell ◽

Marwa M. Afifi ◽

Debasish Paul ◽

Steven D. Cappell

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cell Tracking ◽

Time Lapse ◽

S Phase ◽

Cdk Inhibitor ◽

Cyclin Dependent Kinase ◽

Cell Fates ◽

Damage Response ◽

Time Lapse Imaging

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

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NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2011.09.035 ◽

2011 ◽

Vol 414 (1) ◽

pp. 123-128 ◽

Cited By ~ 8

Author(s):

Burcu Erbaykent-Tepedelen ◽

Besra Özmen ◽

Lokman Varisli ◽

Ceren Gonen-Korkmaz ◽

Bilge Debelec-Butuner ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Cell Lines ◽

Cancer Cell ◽

Dna Damage Response ◽

Prostate Cancer Cell ◽

S Phase ◽

Prostate Cancer Cell Lines ◽

Damage Response ◽

Phase Entry

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Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014048 ◽

2020 ◽

Vol 295 (50) ◽

pp. 17169-17186

Author(s):

Mysore S. Veena ◽

Santanu Raychaudhuri ◽

Saroj K. Basak ◽

Natarajan Venkatesan ◽

Parameet Kumar ◽

...

Keyword(s):

Cell Cycle ◽

Cervical Cancer ◽

Dna Damage ◽

Cell Growth ◽

Cell Lines ◽

Cancer Cell ◽

Dna Damage Response ◽

S Phase ◽

Cervical Cancer Cell ◽

Damage Response

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

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Werner Helicase Control of Human Papillomavirus 16 E1-E2 DNA Replication Is Regulated by SIRT1 Deacetylation

mBio ◽

10.1128/mbio.00263-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 7

Author(s):

Dipon Das ◽

Molly L. Bristol ◽

Nathan W. Smith ◽

Claire D. James ◽

Xu Wang ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Life Cycle ◽

Dna Replication ◽

Homologous Recombination ◽

Dna Damage Response ◽

Human Papillomaviruses ◽

Repair Protein ◽

Damage Response ◽

Dna Repair Protein

ABSTRACTHuman papillomaviruses (HPV) are double-stranded DNA viruses causative in a host of human diseases, including several cancers. Following infection, two viral proteins, E1 and E2, activate viral replication in association with cellular factors and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous recombination (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously, we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2-replicating DNA and regulates the level of replication. Here, we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2-replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2-replicating DNA. We present a model in which E1-E2 replication turns on the DDR, stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2-replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.IMPORTANCEHPV16 is the major viral human carcinogen responsible for between 3 and 4% of all cancers worldwide. Following infection, this virus activates the DNA damage response (DDR) to promote its life cycle and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here, we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall, the results suggest a mechanism by which SIRT1 deacetylation of WRN promotes its interaction with E1-E2-replicating DNA to control the levels and fidelity of that replication.

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