Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair

Global-genomic nucleotide excision repair (GG-NER) is the only pathway available to humans for removal, from the genome overall, of highly genotoxic helix-distorting DNA adducts generated by many environmental mutagens and certain chemotherapeutic agents, e.g., UV-induced 6–4 photoproducts (6–4PPs) and cyclobutane pyrimidine dimers (CPDs). The ataxia telangiectasia and rad-3-related kinase (ATR) is rapidly activated in response to UV-induced replication stress and proceeds to phosphorylate a plethora of downstream effectors that modulate primarily cell cycle checkpoints but also apoptosis and DNA repair. To investigate whether this critical kinase might participate in the regulation of GG-NER, we developed a novel flow cytometry-based DNA repair assay that allows precise evaluation of GG-NER kinetics as a function of cell cycle. Remarkably, inhibition of ATR signaling in primary human lung fibroblasts by treatment with caffeine, or with siRNA specifically targeting ATR, resulted in total inhibition of 6–4PP removal during S phase, whereas cells repaired normally during either G0/G1 or G2/M. Similarly striking S-phase-specific defects in GG-NER of both 6–4PPs and CPDs were documented in ATR-deficient Seckel syndrome skin fibroblasts. Finally, among six diverse model human tumor strains investigated, three manifested complete abrogation of 6–4PP repair exclusively in S-phase populations. Our data reveal a highly novel role for ATR in the regulation of GG-NER uniquely during S phase of the cell cycle, and indicate that many human cancers may be characterized by a defect in this regulation.

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Can vitamin C induce nucleotide excision repair? Support from in vitro evidence

British Journal Of Nutrition ◽

10.1017/s0007114509992285 ◽

2009 ◽

Vol 103 (5) ◽

pp. 686-695 ◽

Cited By ~ 6

Author(s):

Ruth J. Bevan ◽

Nalini Mistry ◽

Parul R. Patel ◽

Eugene P. Halligan ◽

Rosamund Dove ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Stress Response ◽

Vitamin C ◽

Nucleotide Excision Repair ◽

Mononuclear Cells ◽

Culture Supernatant ◽

Excision Repair ◽

Peripheral Blood Mononuclear ◽

Nucleotide Excision

Intracellular vitamin C acts to protect cells against oxidative stress by intercepting reactive oxygen species (ROS) and minimising DNA damage. However, rapid increases in intracellular vitamin C may induce ROS with subsequent DNA damage priming DNA repair processes. Herein, we examine the potential of vitamin C and the derivative ascorbate-2-phosphate (2-AP) to induce a nucleotide excision repair (NER) response to DNA damage in a model of peripheral blood mononuclear cells. Exposure of cells to elevated levels of vitamin C induced ROS activity, resulting in increased levels of deoxycytidine glyoxal (gdC) and 8-oxo-2′-deoxyguanosine (8-oxodG) adducts in DNA; a stress response was also induced by 2-AP, but was delayed in comparison to vitamin C. Evidence of gdC repair was also apparent. Measurement of cyclobutane thymine–thymine dimers (T < >T) in DNA and culture supernatant were included as a positive marker for NER activity; this was evidenced by a reduction in DNA and increases in culture supernatant levels of T < >T for vitamin C-treated cells. Genomics analysis fully supported these findings confirming that 2-AP, in particular, induced genes associated with stress response, cell cycle arrest, DNA repair and apoptosis, and additionally provided evidence for the involvement of vitamin C in the mobilisation of intracellular catalytic Fe.

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Characterization of G1 checkpoint control in the yeast Saccharomyces cerevisiae following exposure to DNA-damaging agents.

Genetics ◽

10.1093/genetics/138.2.271 ◽

1994 ◽

Vol 138 (2) ◽

pp. 271-281 ◽

Cited By ~ 8

Author(s):

W Siede ◽

A S Friedberg ◽

I Dianova ◽

E C Friedberg

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

S Phase ◽

Checkpoint Control ◽

Cycle Arrest ◽

Dna Damaging Agents ◽

Nucleotide Excision ◽

G1 Checkpoint

Abstract The delay of S-phase following treatment of yeast cells with DNA-damaging agents is an actively regulated response that requires functional RAD9 and RAD24 genes. An analysis of cell cycle arrest indicates the existence of (at least) two checkpoints for damaged DNA prior to S-phase; one at START (a G1 checkpoint characterized by pheromone sensitivity of arrested cells) and one between the CDC4- and CDC7-mediated steps (termed the G1/S checkpoint). When a dna1-1 mutant (that affects early events of replicon initiation) also carries a rad9 deletion mutation, it manifests a failure to arrest in G1/S following incubation at the restrictive temperature. This failure to execute regulated G1/S arrest is correlated with enhanced thermosensitivity of colony-forming ability. In an attempt to characterize the signal for RAD9 gene-dependent G1 and G1/S cell cycle arrest, we examined the influence of the continued presence of unexcised photoproducts. In mutants defective in nucleotide excision repair, cessation of S-phase was observed at much lower doses of UV radiation compared to excision-proficient cells. However, this response was not RAD9-dependent. We suggest that an intermediate of nucleotide excision repair, such as DNA strand breaks or single-stranded DNA tracts, is required to activate RAD9-dependent G1 and G1/S checkpoint controls.

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XPA-Mediated Regulation of Global Nucleotide Excision Repair by ATR Is p53-Dependent and Occurs Primarily in S-Phase

PLoS ONE ◽

10.1371/journal.pone.0028326 ◽

2011 ◽

Vol 6 (12) ◽

pp. e28326 ◽

Cited By ~ 27

Author(s):

Zhengke Li ◽

Phillip R. Musich ◽

Moises A. Serrano ◽

Zhiping Dong ◽

Yue Zou

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

S Phase ◽

Nucleotide Excision

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Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1927-1934.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1927-1934

Author(s):

W G McGregor ◽

R H Chen ◽

L Lukash ◽

V M Maher ◽

J J McCormick

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Uv Light ◽

Human Fibroblasts ◽

S Phase ◽

G1 Phase ◽

Strand Bias ◽

Hprt Gene ◽

Significant Difference ◽

Nucleotide Excision

To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.

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Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1927 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1927-1934 ◽

Cited By ~ 115

Author(s):

W G McGregor ◽

R H Chen ◽

L Lukash ◽

V M Maher ◽

J J McCormick

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Uv Light ◽

Human Fibroblasts ◽

S Phase ◽

G1 Phase ◽

Strand Bias ◽

Hprt Gene ◽

Significant Difference ◽

Nucleotide Excision

To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.

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