Detection of DNA Damage

For the detection of free-radical-induced DNA damage and its repair, biochemical techniques are increasingly applied. It would exceed the allocated space to discuss these techniques here, and the reader is referred to the original literature. However, to assist biologists and biophysicists, some of the chemical techniques for measuring typical DNA lesions are discussed. Most of the kinetic data concerning the free-radical chemistry of DNA and its model systems has been obtained by pulse radiolysis. This technique is only available in a few laboratories worldwide. For this reason, it will be described in some detail here. 

In recent years, the sensitivity of the detection of free-radical-induced DNA damage has been considerably increased (for brief reviews, see Cadet and Wein-feld 1993 Cadet et al. 1997a, b, 1999b). There is often a problem with lesions that are refractory to an enzymatic excision (Dizdaroglu et al. 1978, and further examples are reported below), but it is expected that when enzymatic degradation techniques have been further developed, acid hydrolysis and its marked problems will no longer be required. 

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Bprresen, A.-L. (1996) Constant denaturant gel electrophoresis (CDGE) in mutation screening. In Pfeifer, G.P. (ed.) Technologies for Detection of DNA Damage and Mutations. Plenum Press, New York. 

Hachiya N, Sato M, Takizawa Y. 1984. Detection of DNA damage in mutagen-treated mammalian tissues by alkaline elution assay. Mutat Res 130 363. 

McCarroll, N.E., C.E.Piper, and B.H.Keech. 1981. An E. coli microsuspension assay for the detection of DNA damage induced by direct-acting agents and promutagens. Environ. Mutagen. 3 429-444. 

The ATM protein has been identified as an important member of a reaction chain that leads from detection of DNA damage to activation of the p53 protein. Mutations of the ATM protein are causally associated with the disease ataxia telangiectasia, thus the name ATM (ataxia telangiectasia mutated). The ATM protein has protein kinase activity and is counted as a member of the PI3-kinase family, due to sequence homologies (review Canman et al., 1998). The p53 protein is phosphorylated at Serl5 by ATM kinase (Canman et al., 1998) and it is assumed that this phosphorylation contributes to activation of the p53 protein. The ATM protein is preceded by other protein kinases that are directly or indirectly activated by DNA damage and pass this signal on to the p53 protein via the ATM protein. 

Berman, E., House, D.E., Allis, J.W. Simmons, J.E. (1992) Hepatotoxic interactions of ethanol with allyl alcohol or carbon tetrachloride in rats. J. Toxicol, environ. Health, 31, 161-176 Bermudez, E., Mirsalis, J.C. Eales. H.C. (1982) Detection of DNA damage in primary cultures... 

Matsui, S., Yamamoto, R. Yamada, H. (1989) The Bacillus suhtilislm zmiQme, rec-assay for the detection of DNA damaging substances which may occur in chlorinated and ozonated waters. Water Sci. Technol., 21, 875-887... 

The 32P-postlabeling technique allows to improve the sensitivity of the detection of DNA damage (Cadet et al. 1998). The damaged DNA is enzymatically degraded into nucleotide-3-phosphates [reaction (7)]. The resulting mixture of unchanged nucleoside-3-phosphates (dNp) and damaged ones (dXP) are separated by HPLC [reaction (8)]. They are then labeled at the 5 -position with 32P [reaction (9)] and subsequently dephosphorylated at the 3 -position [reaction (10)]. This allows to proceed with a second purification and their identification by, for example, two-dimensional TLC [reactions (11) and (12)]. 

A fluorescent 90-mer oligonucleotide in combination with laser-induced fluorescence and capillary electrophoresis has been used as a versatile probe for a detection of DNA damage (Carnelley et al. 2001). 

M. Fojta and E. Palecek, Supercoiled DNA modified mercury electrode a highly sensitive tool for the detection of DNA damage, Anal. Chim. Acta, 342 (1997) 1-12. 

As we have already mentioned, various methods of immobilization of aptamers onto a solid support are used. In principle these methods are similar to those applied previously for immobilization of single- or double-stranded DNA in genosensors or DNA biosensors for detection of DNA damage (see Pividori et al. [33] for review). The methods of... 

Petzold, G.L., and J.A. Swenberg. Detection of DNA damage induced in vivo following exposure of rats to carcinogens. Cancer Res. 38 1589-1594, 1978. 

Unfortunately, direct electrochemical detection of DNA damage in films suffered from poor signal to noise ratios and data analysis that required derivative or other background corrections. Thus we explored catalytic methods of DNA oxidation (cf. Eqs. 3 and 4) to improve signal to noise in SWV detection.[45] At the same time, we began to realize that layer-by-layer growth of films had... 

Schenkman, J.B. Rusling, J.F. Toxicity screening 28. by electrochemical detection of DNA damage by metabolites generated in-situ in ultrathin DNA-enzyme films. J. Am. Chem. Soc. 2003,125, 1431 —... 

Adams, S.P. Laws, G.M. Storer, R.D. Deluca, J.G. Nichols, W.W. Detection of DNA damage induced by human carcinogens in acellular assays Potential application for determining genotoxic mechanisms. Mutat. Res. 1996, 235 -248. 

Bermudez E, Mirsalis JC, Eales HC (1982) Detection of DNA damage in primary cultures of rat hepatocytes following In Vivo and In Vitro exposure to genotoxic agents. Environ Mutagen 4 667-679... 

Hu QY, Zhu SP. 1990. Detection of DNA damage in spermiogenic stages of mice and treated with enriched uranyl fluoride by alkaline elution. Radiat Environ Biophys 29(3) 161-167. 

Tilby, M. J., Styles, J. M., and Dean, C. J. (1987) Immunological detection of DNA damage caused by melphalan using monoclonal antibodies. Cancer Res. 47, 1542-1546. 

Pavlica, M., Klobucar, G.I., Moja, N., Erben, R. and Pape, D. (2001) Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutat. Res., 490, 209-214. 

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