Radioimmunoassay of DNA Damaged by Ultraviolet Light

Mitchell, David L.

doi:10.1007/978-1-4899-0301-3_6

Cited by 55 publications

(39 citation statements)

References 43 publications

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“…The PBST was decanted, and the blotted membranes were incubated for 10 to 14 h in 2 ml of anti-CTD mouse-derived monoclonal antibodies (Kamiya Biomedical Company, Seattle, Wash.) (29) at an optimized 1:5,000 (wt/vol) dilution in PBST, including 4% membrane blocking agent (Amersham Pharmacia Biotech, Piscataway, N.J.). Incubation was performed in the dark at room temperature (22°C) on an orbital shaker at 150 rpm.…”

Section: Methodsmentioning

confidence: 99%

Photoreactivation in Airborne Mycobacterium parafortuitum

Peccia

Hernandez

2001

Appl Environ Microbiol

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Photoreactivation was observed in airborne Mycobacterium parafortuitum exposed concurrently to UV radiation (254 nm) and visible light. Photoreactivation rates of airborne cells increased with increasing relative humidity (RH) and decreased with increasing UV dose. Under a constant UV dose with visible light absent, the UV inactivation rate of airborne M. parafortuitum cells decreased by a factor of 4 as RH increased from 40 to 95%; however, under identical conditions with visible light present, the UV inactivation rate of airborne cells decreased only by a factor of 2. When irradiated in the absence of visible light, cellular cyclobutane thymine dimer content of UV-irradiated airborne M. parafortuitum and Serratia marcescens increased in response to RH increases. Results suggest that, unlike in waterborne bacteria, cyclobutane thymine dimers are not the most significant form of UV-induced DNA damage incurred by airborne bacteria and that the distribution of DNA photoproducts incorporated into UV-irradiated airborne cells is a function of RH.

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Section: Methodsmentioning

confidence: 99%

Photoreactivation in Airborne Mycobacterium parafortuitum

Peccia

Hernandez

2001

Appl Environ Microbiol

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“…CPDs were quantified using a competitive radioimmunoassay (RIA) as described by Mitchell(1996). The antiserum for CPDs was raised against salmon testes DNA in 20 mM acetophenone (a triplet sensitizer which promotes the production of CPDs but not 6-4 photoproducts) and irradiated with a UV-B source (Westinghouse FS20, 20 W).…”

Section: Methodsmentioning

confidence: 99%

Measurements of DNA damage and photoreactivation imply that most viruses in marine surface waters are infective

Wilhelm¹,

Weinbauer²,

Suttle³

et al. 1998

Aquat. Microb. Ecol.

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The proportion of viruses in natural marine communities that are potentially infectious was inferred from the relationship between DNA damage and the loss of infectivity in marine viral isolates and measurements of the DNA damage in natural viral communities. Several viral isolates which infect marine Vibrio spp. were exposed to UV-C radiation and the concentration of cyclobutane pyrirnidine dimers in the viral DNA was measured with a highly sensitive radioimmunoassay. The loss of infectivity in the UV-exposed isolates was also determined under conditions which either activated or repressed the blue light dependent photolyase enzyme in host cells in order to examine the damagedependent response of this bacterial repair system. In addition, the accumulation of DNA photodamage during the solar day was measured in DNA isolated from natural viral communities collected along a transect in the western Gulf of Mexico. Using the correlation between DNA damage and infectivity for one of the viral isolates, we estimated the proportion of the natural viral community which was infective. The results imply that, due to light-mediated repair of damaged viral DNA by host-cell mechanisms (photoreactivation), greater than 50 % of the viruses in natural communities are infective despite high rates of DNA damage. Furthermore, the accumulation of cyclobutane pyrirnidine dirners was highest at the station where the surface mixed layer was shallowest, emphasizing the importance of mixing depth in relation to the accumulation of DNA damage. These experiments demonstrate that physical parameters such as mixing depth are critically interwoven with light penetration in influencing the infectivity of marine viral communities.

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“…DNA concentration was measured by the fluorometric method (19) with a Hoefer DyNA fluorometer. CPDs and 6-4 products were quantified by a lesion-specific radioimmunoassay (20). Four micrograms of DNA was required for each assay of 6-4 products; one microgram of DNA was used for each assay of CPDs.…”

Section: Methodsmentioning

confidence: 99%

Photorepair mutants of Arabidopsis

Jiang

Yee

Mitchell

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

110

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UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.The biological effects of UV light have been extensively studied in microbes and mammals, where UV irradiation has been shown to have both toxic and mutagenic effects. The two primary UV-induced DNA damage products are the cyclobutane pyrimidine dimer (CPD) and the pyrimidine (6-4) pyrimidinone dimer (the 6-4 product), with 6-4 products making up an estimated 10-25% of all dimers (1). Pyrimidine dimers are known to inhibit microbial and mammalian DNA replication; they have been shown to act in cis to directly inhibit the progress of DNA polymerase, as well as in trans to inhibit the initiation of replication in both microbes and mammals (2, 3). Dimers are, under some circumstances, bypassed in an error-prone fashion, and this trans-lesion synthesis can result in the induction of mutations (4-8). RNA polymerase has also been shown to ''stall'' at both CPDs and 6-4 photoproducts (9-12); in the absence of repair, a single pyrimidine dimer may be sufficient to eliminate expression of a transcriptional unit. Because every pyrimidine dimer acts as a block to transcription and replication, any living tissue, even one in which cell division does not occur, must either avoid or repair UVinduced DNA damage if it is to survive.The CPD and the 6-4 product have very different structures ( Fig. 1) and, therefore, different capacities to form both accurate and inaccurate base pairs (13-15). In recent years the relative contributions of these two lesions to UV-induced cytotoxicity and mutagenicity has been the subject of debate and has been addressed in human and rodent cell lines through a combination of shuttle-vector studies and investigations into the effects of various repair deficiencies. Studies using shuttle vectors may not directly reflect the effects of persisting genomic lesions, because plasmid DNAs lack many of the qualities of genomic DNA, such as high...

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Radioimmunoassay of DNA Damaged by Ultraviolet Light

Cited by 55 publications

References 43 publications

Photoreactivation in Airborne Mycobacterium parafortuitum

Photoreactivation in Airborne Mycobacterium parafortuitum

Measurements of DNA damage and photoreactivation imply that most viruses in marine surface waters are infective

Photorepair mutants of Arabidopsis

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