Background: Dna2 and FEN1 putatively cooperate in Okazaki fragment (OF) processing in yeast. Results: FEN1 depletion leads to OF maturation defects that do not impact replication fork kinetics, whereas hDna2 depletion leads to DNA damage independent of OF maturation. Conclusion: hDna2 participates in DNA replication in a manner distinct from FEN1. Significance: hDna2 participates in replication in a FEN1, OF maturation-independent manner.
We describe a method for synchronization of the cell cycle in the bacterium E. coli. Treatment of asynchronous cultures with the amino acid analog, DL-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, as determined by flow cytometry for DNA content, Southern blotting and microscopy. This method has the advantage that it can be used in fully wild-type cells, at different growth rates, and may be applicable to other bacterial species with replication control by the stringent response. We also elaborate other methods useful for establishing cell cycle parameters in bacterial populations. We describe flow cytometric methods for analyzing bacterial populations for DNA content using the DNA-specific dye PicoGreen, readily detected by most commercial flow cytometers. We also present an method for incorporation of the nucleotide ethynyl-deoxyuridine, EdU, followed by "click" labeling with fluorescent dyes, which allows us to measure and visualize newly replicated DNA in fixed E. coli K-12 cells under non-denaturing conditions.
SummaryThe stringent response is important for bacterial survival under stressful conditions, such as amino acid starvation, and is characterized by the accumulation of ppGpp and pppGpp. ObgE (CgtA, YhbZ) is an essential conserved GTPase in Escherichia coli and several observations have implicated the protein in the control of the stringent response. However, consequences of the protein on specific responses to amino acid starvation have not been noted. We show that ObgE binds to ppGpp with biologically relevant affinity in vitro, implicating ppGpp as an in vivo ligand of ObgE. ObgE mutants increase the ratio of pppGpp to ppGpp within the cell during the stringent response. These changes are correlated with a delayed inhibition of DNA replication by the stringent response, delayed resumption of DNA replication after release, as well as a decreased survival after amino acid deprivation. With these data, we place ObgE as an active effector of the response to amino acid starvation in vivo. Our data correlate the pppGpp/ppGpp ratio with DNA replication control under bacterial starvation conditions, suggesting a possible role for the relative balance of these two nucleotides.
Background:Telomere fragility occurs following replication stress. Results: Leading strand-specific telomere fragility is induced by FEN1 depletion and transcription inhibition and is rescued by ectopic RNase H1 expression. Conclusion: RNA:DNA hybrids contribute to telomere fragility that is limited by FEN1. Significance: This is the first explanation for leading strand-specific telomere fragility and is the first leading strand-specific role for FEN1.
2Age is a significant risk factor for the development of cancer. Both age-dependent 24 accumulation of cell autonomous mutations within preneoplastic cells and increases in 25 senescent stromal cells within the tumor microenvironment are thought to collaborate to 26 drive tumorigenesis. Senescent cells express pro-tumorigenic factors termed the 27 senescence-associated secretory phenotype (SASP), subject to a variety of regulatory 28 mechanisms that are not fully elucidated. Previous work demonstrated that p38 29 mitogen-activated protein kinase (p38MAPK)-dependent regulation of AUF1 occupancy 30 on SASP factor mRNAs post-transcriptionally stabilizes many SASP mRNAs and 31 contributes to their increased expression. Here, we address the mechanism by which 32 p38MAPK regulates AUF1's occupancy and activity on SASP factor mRNAs. We found 33 that the p38MAPK-MK2-HSP27 pathway regulates both mRNA stability and AUF1 34 occupancy in cells induced to senesce. Furthermore, the tumor-promoting abilities of 35 senescent stromal cells were lost upon inhibition of MK2, suggesting that this pathway 36 is a viable therapeutic target within the tumor microenvironment. 37 38 senescence is characterized by an altered cell morphology that includes a flattened 46 3 appearance and development of stress fibers, increased expression of senescence-47 associated β-galactosidase (SA-β-gal), and expression of the senescence-associated 48 secretory phenotype or SASP (also known as the senescence messaging secretome, or 49 SMS) [2][3][4]. This secretory phenotype can lead to an immune-mediated clearance of 50 tumor cells, but when it occurs in stromal cells it functions as a potent tumor promoter 51 [2,[5][6][7]. The SASP consists of a large number of pro-inflammatory cytokines and other 52 immune modulators, matrix remodeling proteins, and pro-proliferative factors, among 53 others, that are coordinately upregulated in senescent cells. Senescent fibroblast-54 derived SASP can promote epithelial cell proliferation and epithelial-to-mesenchymal 55 transition (EMT), to allow for tumor extravasation and metastasis. In addition, the SASP 56 can remodel the stromal compartment to create an immunosuppressive environment 57 conducive to tumor proliferation [2, 7-9]. 58 59 Immediately following a senescence-inducing stimulus, cells (referred to here as pre-60 senescent) begin to upregulate numerous SASP factors through the activity of 61 transcription factors like NFκB but do not yet display classic signs of senescence, 62 including a flattened morphology or SA-β-Gal activity [10][11][12].We recently 63 demonstrated that the maintenance of SASP factor upregulation occurs via a transition 64 from a transcriptionally-driven process in pre-senescent cells to a post-transcriptional 65 stabilization mechanism in cells displaying the morphological changes and SA-β-Gal 66 activity characteristic of senescent cells [10]. In spite of their increased transcriptional 67 levels, several SASP factor mRNAs in pre-senescent cells, including IL-6, IL-8, and GM-68 C...
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