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Cell Cycle (Georgetown, Tex.) 2015Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show...
Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.
Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin-Dependent Kinase 5; DNA Damage; DNA Replication; Female; HeLa Cells; Humans; Hydroxyurea; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Radiation, Ionizing
PubMed: 26237679
DOI: 10.1080/15384101.2015.1078020 -
Journal of Molecular Biology Nov 2013
Topics: DNA Replication; DNA Replication Timing; Genomic Instability; History, 20th Century; History, 21st Century; Molecular Biology; Replicon
PubMed: 24207007
DOI: 10.1016/j.jmb.2013.10.023 -
Cell Cycle (Georgetown, Tex.) 2016The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin...
The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.
Topics: Cell Cycle; Cell Survival; DNA Replication; HCT116 Cells; HeLa Cells; Histone-Lysine N-Methyltransferase; Humans; Repressor Proteins
PubMed: 26771714
DOI: 10.1080/15384101.2015.1121323 -
Biophysical Chemistry Jun 2017Replication stress is a crucial driver of genomic instability. Understanding the mechanisms of replication stress response is instrumental to improve diagnosis and... (Review)
Review
Replication stress is a crucial driver of genomic instability. Understanding the mechanisms of replication stress response is instrumental to improve diagnosis and treatment of human disease. Electron microscopy (EM) is currently the technique of choice to directly visualize a high number of replication intermediates and to monitor their remodeling upon stress. At the same time, DNA fiber analysis is useful to gain mechanistic insight on how genotoxic agents perturb replication fork dynamics genome-wide at single-molecule resolution. Combining these techniques has proven invaluable to achieve a comprehensive view of the mechanisms that ensure error-free processing of damaged replication forks. Here, we review how EM and single-molecule DNA fiber approaches can be used together to shed light into the mechanisms of replication stress response and discuss important cautions to be taken into account when comparing results obtained by EM and DNA fiber.
Topics: DNA Damage; DNA Replication; Genomic Instability; Humans; Microscopy, Electron; Mutagens; Single Molecule Imaging
PubMed: 27939387
DOI: 10.1016/j.bpc.2016.11.014 -
ACS Synthetic Biology Jul 2018The yeast cytoplasmically localized pGKL1/TP-DNAP1 plasmid/DNA polymerase pair forms an orthogonal DNA replication system whose mutation rate can be drastically...
The yeast cytoplasmically localized pGKL1/TP-DNAP1 plasmid/DNA polymerase pair forms an orthogonal DNA replication system whose mutation rate can be drastically increased without influencing genomic replication, thereby supporting in vivo continuous evolution. Here, we report that the pGKL2/TP-DNAP2 plasmid/DNA polymerase pair forms a second orthogonal replication system. We show that custom genes can be encoded and expressed from pGKL2, that error-prone TP-DNAP2s can be engineered, and that pGKL2 replication by TP-DNAP2 is both orthogonal to genomic replication in Saccharomyces cerevisiae and mutually orthogonal with pGKL1 replication by TP-DNAP1. This demonstration of two mutually orthogonal DNA replication systems with tunable error rates and properties should enable new applications in cell-based continuous evolution, genetic recording, and synthetic biology at large.
Topics: DNA Replication; DNA-Directed DNA Polymerase; Metabolic Engineering; Plasmids
PubMed: 29969238
DOI: 10.1021/acssynbio.8b00195 -
Endocrinology Jul 2009Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high, and exposure to progesterone...
Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high, and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk, yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast, we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing, including E2F transcription factors, chromatin licensing and DNA replication factor 1 (Cdt1), and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone, showing that progesterone influences epithelial cell lineage differentiation. Therefore, we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers.
Topics: Breast; Cell Differentiation; Cell Proliferation; Cells, Cultured; DNA Replication; Epithelial Cells; Female; Humans; Progesterone; Receptors, Estrogen; Receptors, Progesterone; Stem Cells
PubMed: 19342456
DOI: 10.1210/en.2008-1630 -
Plant Signaling & Behavior Apr 2017Recent advances in replicative DNA labeling technology have allowed new ways to study DNA replication in living plants. Temporal and spatial aspects of DNA replication...
Recent advances in replicative DNA labeling technology have allowed new ways to study DNA replication in living plants. Temporal and spatial aspects of DNA replication programs are believed to derive from genomic structure and function. Bass et al. (2015) recently visualized DNA synthesis using 3D microscopy of nuclei at three sub-stages of S phase: early, middle and late. This addendum expands on that study by comparing plant and animal DNA replication patterns, by considering implications of the two-compartment model of euchromatin, and by exploring the meaning of the DNA labeling signals inside the nucleolus. Finally, we invite the public to explore and utilize 300 image data sets through OMERO, a teaching and research web resource for visualization, management, or analysis of microscopic data.
Topics: Chromatin; DNA Replication; DNA, Plant; DNA, Ribosomal; Zea mays
PubMed: 28375043
DOI: 10.1080/15592324.2017.1311437 -
BioEssays : News and Reviews in... Jul 2023Despite advances in treatments over the last decades, a uniformly reliable and free of side effects therapy of human cancers remains to be achieved. During chromosome...
Despite advances in treatments over the last decades, a uniformly reliable and free of side effects therapy of human cancers remains to be achieved. During chromosome replication, a premature halt of two converging DNA replication forks would cause incomplete replication and a cytotoxic chromosome nondisjunction during mitosis. In contrast to normal cells, most cancer cells bear numerous DNA deletions. A homozygous deletion permanently marks a cell and its descendants. Here, we propose an approach to cancer therapy in which a pair of sequence-specific roadblocks is placed solely at two cancer-confined deletion sites that are located ahead of two converging replication forks. We describe this method, termed "replication blocks specific for deletions" (RBSD), and another deletions-based approach as well. RBSD can be expanded by placing pairs of replication roadblocks on several different chromosomes. The resulting simultaneous nondisjunctions of these chromosomes in cancer cells would further increase the cancer-specific toxicity of RBSD.
Topics: Humans; Homozygote; Sequence Deletion; DNA; DNA Replication; Neoplasms
PubMed: 37166062
DOI: 10.1002/bies.202300051 -
Virology Mar 2010Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori)....
Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gene located in cis to the ori. Under optimized conditions, replication of the SV40 and HPV31 ori-plasmids resulted in a 50- and 150-fold increase in firefly luciferase levels, respectively. These results were validated using replication-defective mutants of LT, E1 and E2 and with inhibitors of DNA replication and cell-cycle progression. These quantitative and high-throughput assays should greatly facilitate the study of SV40 and HPV31 DNA replication and the identification of small-molecule inhibitors of this process.
Topics: Antigens, Viral, Tumor; Antiviral Agents; Blotting, Southern; Blotting, Western; Cell Line, Tumor; DNA Replication; Deoxycytidine; Fluorescence Polarization; High-Throughput Screening Assays; Humans; Luciferases; Papillomaviridae; Polyomavirus; Reverse Transcriptase Polymerase Chain Reaction; Simian virus 40; Virus Replication; Gemcitabine
PubMed: 20079917
DOI: 10.1016/j.virol.2009.12.026 -
Annual Review of Medicine 2008Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss... (Review)
Review
Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA. This review focuses on our current understanding of nuclear gene mutations that produce mtDNA alterations and cause mitochondrial depletion syndrome (MDS), progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). To date, all of these etiologic nuclear genes fall into one of two categories: genes whose products function directly at the mtDNA replication fork, such as POLG, POLG2, and TWINKLE, or genes whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly the newly identified MPV17.
Topics: DNA Replication; DNA, Mitochondrial; Humans; Mitochondrial Diseases; Mutation
PubMed: 17892433
DOI: 10.1146/annurev.med.59.053006.104646