Replication Past the -Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase

-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replicationin vitroand is mutagenic in mammalian cells. Herein we reportin vitrotranslesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase (hPol and yPol ). dAMP misincorporation opposite the cross-linked G by yPol was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for :A compared to :C. For hPol , both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol was more error-prone. Mutational frequencies of yPol and hPol were 36% and 14%, respectively. Targeted was the dominant mutation by both DNA polymerases. But yPol induced targeted in 23% frequency relative to 4% by hPol . For yPol , targeted and constituted 83% of the mutations. By contrast, with hPol , semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells.

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Mutational Specificity of γ-Radiation-Induced Guanine−Thymine and Thymine−Guanine Intrastrand Cross-Links in Mammalian Cells and Translesion Synthesis Past the Guanine−Thymine Lesion by Human DNA Polymerase η†

Biochemistry ◽

10.1021/bi800529f ◽

2008 ◽

Vol 47 (31) ◽

pp. 8070-8079 ◽

Cited By ~ 43

Author(s):

Laureen C. Colis ◽

Paromita Raychaudhury ◽

Ashis K. Basu

Keyword(s):

Dna Polymerase ◽

Mammalian Cells ◽

Translesion Synthesis ◽

Γ Radiation ◽

Human Dna ◽

Mutational Specificity ◽

Radiation Induced ◽

Cross Links

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DNA polymerase ζ in DNA replication and repair

Nucleic Acids Research ◽

10.1093/nar/gkz705 ◽

2019 ◽

Vol 47 (16) ◽

pp. 8348-8361 ◽

Cited By ~ 14

Author(s):

Sara K Martin ◽

Richard D Wood

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Dna Sequences ◽

Mammalian Cells ◽

Translesion Synthesis ◽

Dna Lesions ◽

Replication Forks ◽

Dna Replication And Repair ◽

Radiation Induced ◽

Break Induced Replication

Abstract Here, we survey the diverse functions of DNA polymerase ζ (pol ζ) in eukaryotes. In mammalian cells, REV3L (3130 residues) is the largest catalytic subunit of the DNA polymerases. The orthologous subunit in yeast is Rev3p. Pol ζ also includes REV7 subunits (encoded by Rev7 in yeast and MAD2L2 in mammalian cells) and two subunits shared with the replicative DNA polymerase, pol δ. Pol ζ is used in response to circumstances that stall DNA replication forks in both yeast and mammalian cells. The best-examined situation is translesion synthesis at sites of covalent DNA lesions such as UV radiation-induced photoproducts. We also highlight recent evidence that uncovers various roles of pol ζ that extend beyond translesion synthesis. For instance, pol ζ is also employed when the replisome operates sub-optimally or at difficult-to-replicate DNA sequences. Pol ζ also participates in repair by microhomology mediated break-induced replication. A rev3 deletion is tolerated in yeast but Rev3l disruption results in embryonic lethality in mice. Inactivation of mammalian Rev3l results in genomic instability and invokes cell death and senescence programs. Targeting of pol ζ function may be a useful strategy in cancer therapy, although chromosomal instability associated with pol ζ deficiency must be considered.

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Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in vitro

Journal of Cell Science ◽

10.1242/jcs.94.3.517 ◽

1989 ◽

Vol 94 (3) ◽

pp. 517-525

Author(s):

A.M. Mes-Masson ◽

S. Masson ◽

D. Banville ◽

L. Chalifour

Keyword(s):

Mammalian Cells ◽

Mouse Kidney ◽

Kidney Cells ◽

T Antigens ◽

Primary Mouse ◽

Specific Antisera ◽

Growth Properties ◽

Metallothionein Promoter ◽

Specific Mrna

A recombinant plasmid (pMTONCO) containing the coding sequences for rat oncomodulin under the direction of the metallothionein promoter was constructed. pMTONCO was co-transfected with the pSV2-NEO plasmid into primary mouse kidney cells or Rat-1 cells using the calcium phosphate technique and stable transformants were isolated after selection with G418. Transcription from the metallothionein promoter was inducible with heavy metals and produced an oncomodulin-specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immunoprecipitation with specific antisera. While a plasmid encoding the polyomavirus T-antigens was able to prolong the life-span of primary mouse kidney cells in culture, no equivalent activity was noted when the pMTONCO plasmid was used to transfect primary cells. When expressed in Rat-1 cells, oncomodulin did not affect the growth properties of these cells, nor did it predispose cells to higher frequencies of oncogenic transformation to a viral oncogene. We conclude that oncomodulin is neither an immortalizing nor transforming agent in vitro.

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Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells

Biochemical Journal ◽

10.1042/bj20050657 ◽

2005 ◽

Vol 391 (1) ◽

pp. 33-40 ◽

Cited By ~ 66

Author(s):

Nedjma Zidi-Yahiaoui ◽

Isabelle Mouro-Chanteloup ◽

Anne-Marie D'Ambrosio ◽

Claude Lopez ◽

Pierre Gane ◽

...

Keyword(s):

Mammalian Cells ◽

Ammonium Transport ◽

Kidney Cells ◽

Wild Type ◽

Kinetic Rate Constant ◽

Kinetic Rate ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Ph Variation ◽

Rh Glycoproteins

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin–Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 μM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222–17227] constitute a family of NH3 channels in mammalian cells.

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Synthesis in vitro of urokinase-like material using polyA(+)RNA from human kidney and from cultured human embryonic kidney cells

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(80)80155-0 ◽

1980 ◽

Vol 97 (1) ◽

pp. 207-215 ◽

Cited By ~ 6

Author(s):

A. Bollen ◽

C. Glineur ◽

A. Herzog

Keyword(s):

Human Kidney ◽

Kidney Cells ◽

Human Embryonic Kidney ◽

Human Embryonic Kidney Cells

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A Role for DNA Polymerase β in Mutagenic UV Lesion Bypass

Journal of Biological Chemistry ◽

10.1074/jbc.m207101200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 50046-50053 ◽

Cited By ~ 48

Author(s):

Laurence Servant ◽

Christophe Cazaux ◽

Anne Bieth ◽

Shigenori Iwai ◽

Fumio Hanaoka ◽

...

Keyword(s):

Dna Polymerase ◽

Genetic Instability ◽

Mammalian Cells ◽

Excision Repair ◽

Pyrimidine Dimer ◽

Dna Polymerase Β ◽

Lesion Bypass ◽

Base Excision

We report here that DNA polymerase β (pol β), the base excision repair polymerase, is highly expressed in human melanoma tissues, known to be associated with UV radiation exposure. To investigate the potential role of pol β in UV-induced genetic instability, we analyzed the cellular and molecular effects of excess pol β. We firstly demonstrated that mammalian cells overexpressing pol β are resistant and hypermutagenic after UV irradiation and that replicative extracts from these cells are able to catalyze complete translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). By usingin vitroprimer extension reactions with purified pol β, we showed that CPD as well as, to a lesser extent, the thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct, were bypassed. pol β mostly incorporates the correct dATP opposite the 3′-terminus of both CPD and the (6-4) photoproduct but can also misinsert dCTP at a frequency of 32 and 26%, respectively. In the case of CPD, efficient and error-prone extension of the correct dATP was found. These data support a biological role of pol β in UV lesion bypass and suggest that deregulated pol β may enhance UV-induced genetic instability.

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Induction of Sertoli cells from human fibroblasts by NR5A1 and GATA4

10.1101/430900 ◽

2018 ◽

Author(s):

Jianlin Liang ◽

Nan Wang ◽

Jing He ◽

Jian Du ◽

Yahui Guo ◽

...

Keyword(s):

Sertoli Cells ◽

Lipid Droplets ◽

Human Fibroblasts ◽

Seminiferous Tubules ◽

Kidney Cells ◽

Human Embryonic Kidney ◽

Human Embryonic Kidney Cells ◽

Reprogramming Factors ◽

Spermatogonia Cells

AbstractSertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). The induction of human Sertoli cells from fibroblasts could provide cellular sources for fertility and transplantation treatments. Here, we report the in vitro reprogramming of human fibroblasts to Sertoli cells and characterize these human induced Sertoli cells (hiSCs). Initially, five transcriptional factors (NR5A1, GATA4, WT1, SOX9 and DMRT1) and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, i.e., NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles that are similar to those of primary human Sertoli cells. Consistent with the known cellular properties of Sertoli cells, hiSCs attract endothelial cells and exhibit high number of lipid droplets in the cytoplasm. More importantly, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. In addition, hiSCs suppress the production of IL-2 and proliferation of human T lymphocytes. When hiSCs were cotransplanted with human embryonic kidney cells, these xenotransplanted human cells survived longer in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli-only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.

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Transient DNA Double-Strand Breakage in 4-Hydroperoxyifosfamide-Treated Mammalian Cells in Vitro Does Not Interact with the Rejoining of Radiation-Induced Double-Strand Breaks

Strahlentherapie und Onkologie ◽

10.1007/s00066-002-0929-4 ◽

2002 ◽

Vol 178 (5) ◽

pp. 269-274 ◽

Cited By ~ 1

Author(s):

Detlev Latz ◽

Klaus-Josef Weber

Keyword(s):

Mammalian Cells ◽

Double Strand Breaks ◽

Strand Breaks ◽

Radiation Induced ◽

Strand Breakage

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A Scanning Electron-Microscope Study of the Surface Features of Mammalian Cells In Vitro

Journal of Cell Science ◽

10.1242/jcs.11.1.233 ◽

1972 ◽

Vol 11 (1) ◽

pp. 233-247

Author(s):

GISELE M. HODGES ◽

MARJORIE D. MUIR

Keyword(s):

Cell Surface ◽

Mammalian Cells ◽

Physiological State ◽

Culture Conditions ◽

Kidney Cells ◽

Scanning Electron Microscope Study ◽

Cell Strains ◽

Scanning Electron ◽

Morphology Of Surface

The cell surface aspects and the density, distribution and morphology of surface cytoplasmic projections (microprocesses) of HeLa, BHK and baby mouse kidney cells, maintained under different culture conditions, have been compared using scanning electron microscopy. No significant differences were noted in the surface aspects of these cell strains. Comparison of the cell strains, under identical culture conditions, showed variations from one strain to another in the density and morphology of the cell surface microprocesses which may reflect basic physiological differences between the strains. For a given cell strain, variations were observed in response to changes in culture conditions indicative that the presence of surface microprocesses is related to the physiological state of the cell.

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