Inverse correlation between p53 protein levels and DNA repair efficiency in human fibroblast strains treated with 4-nitroquinoline 1-oxide: evidence that lesions other than DNA strand breaks trigger the p53 response

The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.

Download Full-text

Lack of Correlation Between DNA Strand Breakage and p53 Protein Levels in Human Fibroblast Strains Exposed to Ultraviolet Light¶

Photochemistry and Photobiology ◽

10.1562/0031-8655(2000)072<0562:locbds>2.0.co;2 ◽

2000 ◽

Vol 72 (4) ◽

pp. 562 ◽

Cited By ~ 2

Author(s):

Louise Enns ◽

David Murray ◽

Razmik Mirzayans

Keyword(s):

Ultraviolet Light ◽

Human Fibroblast ◽

P53 Protein ◽

Protein Levels ◽

Strand Breakage ◽

Human Fibroblast Strains ◽

Dna Strand ◽

Dna Strand Breakage

Download Full-text

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.6.l1174 ◽

1997 ◽

Vol 272 (6) ◽

pp. L1174-L1180 ◽

Cited By ~ 25

Author(s):

M. Takeoka ◽

W. F. Ward ◽

H. Pollack ◽

D. W. Kamp ◽

R. J. Panos

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Epithelial Cells ◽

Dna Polymerase ◽

Dna Polymerases ◽

Dna Strand Breaks ◽

Strand Breaks ◽

Alveolar Epithelial ◽

Radiation Induced ◽

Dna Strand

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

Download Full-text

Influence of Acute Exercise on DNA Repair and PARP Activity before and after Irradiation in Lymphocytes from Trained and Untrained Individuals

International Journal of Molecular Sciences ◽

10.3390/ijms20122999 ◽

2019 ◽

Vol 20 (12) ◽

pp. 2999 ◽

Cited By ~ 5

Author(s):

Maria Moreno-Villanueva ◽

Andreas Kramer ◽

Tabea Hammes ◽

Maria Venegas-Carro ◽

Patrick Thumm ◽

...

Keyword(s):

Physical Activity ◽

Dna Damage ◽

Dna Repair ◽

Immune Cells ◽

Acute Exercise ◽

Dna Strand Breaks ◽

Strand Breaks ◽

Before And After ◽

Radiation Induced ◽

Dna Strand

Several studies indicate that acute exercise induces DNA damage, whereas regular exercise increases DNA repair kinetics. Although the molecular mechanisms are not completely understood, the induction of endogenous reactive oxygen species (ROS) during acute exhaustive exercise due to metabolic processes might be responsible for the observed DNA damage, while an adaptive increase in antioxidant capacity due to regular physical activity seems to play an important protective role. However, the protective effect of physical activity on exogenously induced DNA damage in human immune cells has been poorly investigated. We asked the question whether individuals with a high aerobic capacity would have an enhanced response to radiation-induced DNA damage. Immune cells are highly sensitive to radiation and exercise affects lymphocyte dynamics and immune function. Therefore, we measured endogenous and radiation-induced DNA strand breaks and poly (ADP-ribose) polymerase-1 (PARP1) activity in peripheral blood mononuclear cells (PBMCs) from endurance-trained (maximum rate of oxygen consumption measured during incremental exercise V’O2max > 55 mL/min/kg) and untrained (V’O2max < 45 mL/min/kg) young healthy male volunteers before and after exhaustive exercise. Our results indicate that: (i) acute exercise induces DNA strand breaks in lymphocytes only in untrained individuals, (ii) following acute exercise, trained individuals repaired radiation-induced DNA strand breaks faster than untrained individuals, and (iii) trained subjects retained a higher level of radiation-induced PARP1 activity after acute exercise. The results of the present study indicate that increased aerobic fitness can protect immune cells against radiation-induced DNA strand breaks.

Download Full-text

Mechanistic insight into the role of Poly(ADP-ribosyl)ation in DNA topology modulation and response to DNA damage

Mutagenesis ◽

10.1093/mutage/gez045 ◽

2019 ◽

Vol 35 (1) ◽

pp. 107-118

Author(s):

Bakhyt T Matkarimov ◽

Dmitry O Zharkov ◽

Murat K Saparbaev

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genotoxic Stress ◽

Strand Break ◽

Dna Supercoiling ◽

Dna Strand Breaks ◽

Chromosomal Dna ◽

Dna Breaks ◽

Strand Breaks ◽

Dna Strand

Abstract Genotoxic stress generates single- and double-strand DNA breaks either through direct damage by reactive oxygen species or as intermediates of DNA repair. Failure to detect and repair DNA strand breaks leads to deleterious consequences such as chromosomal aberrations, genomic instability and cell death. DNA strand breaks disrupt the superhelical state of cellular DNA, which further disturbs the chromatin architecture and gene activity regulation. Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyse the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are regarded as DNA damage sensors that, upon activation by strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. Noteworthy, the regularly branched structure of poly(ADP-ribose) polymer suggests that the mechanism of its synthesis may involve circular movement of PARP1 around the DNA helix, with a branching point in PAR corresponding to one complete 360° turn. We propose that PARP1 stays bound to a DNA strand break end, but rotates around the helix displaced by the growing poly(ADP-ribose) chain, and that this rotation could introduce positive supercoils into damaged chromosomal DNA. This topology modulation would enable nucleosome displacement and chromatin decondensation around the lesion site, facilitating the access of DNA repair proteins or transcription factors. PARP1-mediated DNA supercoiling can be transmitted over long distances, resulting in changes in the high-order chromatin structures. The available structures of PARP1 are consistent with the strand break-induced PAR synthesis as a driving force for PARP1 rotation around the DNA axis.

Download Full-text

DNA Repair Functions That Control Sensitivity to Topoisomerase-Targeting Drugs

Eukaryotic Cell ◽

10.1128/ec.3.1.82-90.2004 ◽

2004 ◽

Vol 3 (1) ◽

pp. 82-90 ◽

Cited By ~ 39

Author(s):

Mobeen Malik ◽

John L. Nitiss

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Topoisomerase I ◽

Topoisomerase Ii ◽

Excision Repair ◽

Dna Strand Breaks ◽

Dna Topology ◽

Strand Breaks ◽

Wide Range ◽

Dna Strand

ABSTRACT DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation. Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate. Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents. Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs. We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment. S. pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II. In contrast to results observed with Saccharomyces cerevisiae, S. pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents. The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors. Finally, repair genes (such as the S. pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs. Since the pattern of sensitivity is distinct from that seen with other systems (such as the S. cerevisiae system), our results highlight the usefulness of S. pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases.

Download Full-text

Seasonal variation in mussel Mytilus edulis digestive gland cytochrome P4501A- and 2E-immunoidentified protein levels and DNA strand breaks (Comet assay)

Marine Environmental Research ◽

10.1016/s0141-1136(00)00040-4 ◽

2000 ◽

Vol 50 (1-5) ◽

pp. 405-409 ◽

Cited By ~ 27

Author(s):

J.P Shaw ◽

A.T Large ◽

J.K Chipman ◽

D.R Livingstone ◽

L.D Peters

Keyword(s):

Seasonal Variation ◽

Comet Assay ◽

Mytilus Edulis ◽

Digestive Gland ◽

Dna Strand Breaks ◽

Strand Breaks ◽

Protein Levels ◽

Cytochrome P4501a ◽

Dna Strand

Download Full-text

ARF induction in response to DNA strand breaks is regulated by PARP1

Nucleic Acids Research ◽

10.1093/nar/gkt1185 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2320-2329 ◽

Cited By ~ 19

Author(s):

Giulia Orlando ◽

Svetlana V. Khoronenkova ◽

Irina I. Dianova ◽

Jason L. Parsons ◽

Grigory L. Dianov

Keyword(s):

Dna Damage ◽

Protein Function ◽

P53 Protein ◽

Dna Strand Breaks ◽

Vital Role ◽

Cellular Stress Response ◽

Strand Breaks ◽

Dna Strand ◽

Cellular Dna ◽

Arf Protein

Abstract The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating up-regulation of p53 protein and consequently promoting cell-cycle delay. Although p53 protein function has been clearly linked to the cellular DNA damage response, the role of ARF protein in this process is unclear. Here, we report that arf gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage. We discovered that poly(ADP-ribose) synthesis catalysed by PARP1 at the sites of unrepaired SBs activates ARF transcription through a protein signalling cascade, including the NAD+-dependent deacetylase SIRT1 and the transcription factor E2F1. Our data suggest that poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD+, thus down-regulating SIRT1 activity and consequently activating E2F1-dependent ARF transcription. Our findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.

Download Full-text