Validation of the nucleotide excision repair comet assay on cryopreserved PBMCs to measure inter-individual variation in DNA repair capacity

Abstract Introduction Veterans of the 1991 Gulf War were potentially exposed to a mixture of stress, chemicals and radiation that may have contributed to the persistent symptoms of Gulf War Illness (GWI). The genotoxic effects of some of these exposures are mediated by the DNA nucleotide excision repair (NER) pathway. We hypothesized that individuals with relatively low DNA repair capacity would suffer greater damage from cumulative genotoxic exposures, some of which would persist, causing ongoing problems. Materials and Methods Blood samples were obtained from symptomatic Gulf War veterans and age-matched controls. The unscheduled DNA synthesis assay, a functional measurement of NER capacity, was performed on cultured lymphocytes, and lymphocyte mRNA was extracted and analyzed by sequencing. Results Despite our hypothesis that GWI would be associated with DNA repair deficiency, NER capacity in lymphocytes from affected GWI veterans actually exhibited a significantly elevated level of DNA repair (p = 0.016). Both total gene expression and NER gene expression successfully differentiated individuals with GWI from unaffected controls. The observed functional increase in DNA repair capacity was accompanied by an overexpression of genes in the NER pathway, as determined by RNA sequencing analysis. Conclusion We suggest that the observed elevations in DNA repair capacity and NER gene expression are indicative of a “hormetic,” i.e., induced or adaptive protective response to battlefield exposures. Normally such effects are short-term, but in these individuals this response has resulted in a long-term metabolic shift that may also be responsible for the persistent symptoms of GWI.

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An Xpb Mouse Model for Combined Xeroderma Pigmentosum and Cockayne Syndrome Reveals Progeroid Features upon Further Attenuation of DNA Repair

Molecular and Cellular Biology ◽

10.1128/mcb.01229-08 ◽

2008 ◽

Vol 29 (5) ◽

pp. 1276-1290 ◽

Cited By ~ 39

Author(s):

Jaan-Olle Andressoo ◽

Geert Weeda ◽

Jan de Wit ◽

James R. Mitchell ◽

Rudolf B. Beems ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Xeroderma Pigmentosum ◽

Molecular Mechanisms ◽

Excision Repair ◽

Cockayne Syndrome ◽

Repair Capacity ◽

Dna Repair Capacity ◽

Neonatal Lethality ◽

Nucleotide Excision

ABSTRACT Patients carrying mutations in the XPB helicase subunit of the basal transcription and nucleotide excision repair (NER) factor TFIIH display the combined cancer and developmental-progeroid disorder xeroderma pigmentosum/Cockayne syndrome (XPCS). Due to the dual transcription repair role of XPB and the absence of animal models, the underlying molecular mechanisms of XPBXPCS are largely uncharacterized. Here we show that severe alterations in Xpb cause embryonic lethality and that knock-in mice closely mimicking an XPCS patient-derived XPB mutation recapitulate the UV sensitivity typical for XP but fail to show overt CS features unless the DNA repair capacity is further challenged by crossings to the NER-deficient Xpa background. Interestingly, the Xpb XPCS Xpa double mutants display a remarkable interanimal variance, which points to stochastic DNA damage accumulation as an important determinant of clinical diversity in NER syndromes. Furthermore, mice carrying the Xpb XPCS mutation together with a point mutation in the second TFIIH helicase Xpd are healthy at birth but display neonatal lethality, indicating that transcription efficiency is sufficient to permit embryonal development even when both TFIIH helicases are crippled. The double-mutant cells exhibit sensitivity to oxidative stress, suggesting a role for endogenous DNA damage in the onset of XPB-associated CS.

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An XPG DNA repair defect causing mutagen hypersensitivity in mouse leukemia L1210 cells.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.290 ◽

1995 ◽

Vol 15 (1) ◽

pp. 290-297 ◽

Cited By ~ 20

Author(s):

J A Vilpo ◽

L M Vilpo ◽

D E Szymkowski ◽

A O'Donovan ◽

R D Wood

Keyword(s):

Dna Repair ◽

Nucleotide Excision Repair ◽

Cisplatin Resistance ◽

Excision Repair ◽

Model Systems ◽

Repair Capacity ◽

Mouse Leukemia ◽

L1210 Cells ◽

Cell Extracts ◽

Nucleotide Excision

One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.

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Evidence for the Involvement of DNA Repair Enzyme NEIL1 in Nucleotide Excision Repair of (5′R)- and (5′S)-8,5′-Cyclo-2′-deoxyadenosines

Biochemistry ◽

10.1021/bi902161f ◽

2010 ◽

Vol 49 (6) ◽

pp. 1053-1055 ◽

Cited By ~ 41

Author(s):

Pawel Jaruga ◽

Yan Xiao ◽

Vladimir Vartanian ◽

R. Stephen Lloyd ◽

Miral Dizdaroglu

Keyword(s):

Dna Repair ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Repair Enzyme ◽

Nucleotide Excision

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Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3041-3049.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3041-3049

Author(s):

L Bardwell ◽

A J Cooper ◽

E C Friedberg

Keyword(s):

Dna Repair ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Specific Interaction ◽

Mitotic Recombination ◽

Nucleotide Excision ◽

Specific Association ◽

Recombination Pathway ◽

Cloned Gene

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are two of at least seven genes which are known to be required for damage-specific recognition and/or damage-specific incision of DNA during nucleotide excision repair. RAD1 and RAD10 are also involved in a specialized mitotic recombination pathway. We have previously reported the purification of the RAD10 protein to homogeneity (L. Bardwell, H. Burtscher, W. A. Weiss, C. M. Nicolet, and E. C. Friedberg, Biochemistry 29:3119-3126, 1990). In the present studies we show that the RAD1 protein, produced by in vitro transcription and translation of the cloned gene, specifically coimmunoprecipitates with the RAD10 protein translated in vitro or purified from yeast. Conversely, in vitro-translated RAD10 protein specifically coimmunoprecipitates with the RAD1 protein. The sites of this stable and specific interaction have been mapped to the C-terminal regions of both polypeptides. This portion of RAD10 protein is evolutionarily conserved. These results are the first biochemical evidence of a specific association between any eukaryotic proteins genetically identified as belonging to a recombination or DNA repair pathway and suggest that the RAD1 and RAD10 proteins act at the same or consecutive biochemical steps in both nucleotide excision repair and mitotic recombination.

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Structural basis of TFIIH activation for nucleotide excision repair

10.1101/628032 ◽

2019 ◽

Author(s):

Goran Kokic ◽

Aleksandar Chernev ◽

Dimitry Tegunov ◽

Christian Dienemann ◽

Henning Urlaub ◽

...

Keyword(s):

Dna Repair ◽

Transcription Factor ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Dna Lesions ◽

Structural Basis ◽

Disease Mutations ◽

Mechanistic Analysis ◽

Nucleotide Excision ◽

Dna Complex

AbstractGenomes are constantly threatened by DNA damage, but cells can remove a large variety of DNA lesions by nucleotide excision repair (NER)1. Mutations in NER factors compromise cellular fitness and cause human diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome and trichothiodystrophy2,3. The NER machinery is built around the multisubunit transcription factor IIH (TFIIH), which opens the DNA repair bubble, scans for the lesion, and coordinates excision of the damaged DNA single strand fragment1,4. TFIIH consists of a kinase module and a core module that contains the ATPases XPB and XPD5. Here we prepare recombinant human TFIIH and show that XPB and XPD are stimulated by the additional NER factors XPA and XPG, respectively. We then determine the cryo-electron microscopy structure of the human core TFIIH-XPA-DNA complex at 3.6 Å resolution. The structure represents the lesion-scanning intermediate on the NER pathway and rationalizes the distinct phenotypes of disease mutations. It reveals that XPB and XPD bind double- and single-stranded DNA, respectively, consistent with their translocase and helicase activities. XPA forms a bridge between XPB and XPD, and retains the DNA at the 5’-edge of the repair bubble. Biochemical data and comparisons with prior structures6,7 explain how XPA and XPG can switch TFIIH from a transcription factor to a DNA repair factor. During transcription, the kinase module inhibits the repair helicase XPD8. For DNA repair, XPA dramatically rearranges the core TFIIH structure, which reorients the ATPases, releases the kinase module and displaces a ‘plug’ element from the DNA-binding pore in XPD. This enables XPD to move by ~80 Å, engage with DNA, and scan for the lesion in a XPG-stimulated manner. Our results provide the basis for a detailed mechanistic analysis of the NER mechanism.

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