Overview of techniques

Polymerase-based DNA end-labeling assays have been used for decades to detect DNA breaks. The basic concept is that labeled nucleotides are added to gaps or nicks in one of the DNA strands and to recessing strands at the double-strand break. A reaction mixture containing labeled nucleotides and appropriate enzymes is applied to permeabilized tissue culture cells or tissue sections. The enzymes that are used can catalyze polymerization of added nucleotides without a single-strand DNA template. A variety of methods are used to detect the labels; detailed descriptions of them are published elsewhere (Didenko, 2002). Terminal transferase, Escherichia coli DNA polymerase (Pol I), and the "Klenow fragment'' are the polymerases used most often in the end-labeling reactions. The different modes of action of these enzymes can provide information about the nature of DNA breaks.

Pol I binds to DNA at a single-strand break ("nick") with a 3'-OH terminus. Replication starts by covalent extension of the 3'-OH terminus. The enzyme has concurrent 5'-3' nuclease and polymerase activity. A primer strand is constantly uncovered by the nuclease activity by hydrolysis at the 50 side of the nick, while the polymerase activity catalyses the addition of nucleotides to the 30 end. The net result of simultaneous hydrolysis and synthesis is translation of the nick along the DNA duplex in the 5'-3' direction, incorporating labeled nucleotides (Kelly et al., 1970; Manoharan et al., 1987; Nose and Okamoto, 1983; Snyder and Matheson, 1985). Cleavage of Pol I by subtilisin creates fragments of 76 and 34 kDa. The 76-kDa fragment has polymerizing but not 50!30 nuclease activity. Following its discovery by Klenow and Henningsen (1970), the large fragment has been called the "Klenow fragment." In order to polymerize the addition of nucleotides to the 30-OH end of DNA, the Klenow fragment requires a single-stranded DNA template and a DNA or RNA primer with a 30-OH terminus. The ability of the Klenow fragment to label DNA with a 50 overhang is used to detect DNA breaks in situ (reviewed in Wood, 2002). Terminal deoxynucleotidyl transferase (TdT) is another enzyme often used for DNA end labeling (reviewed in Loo, 2002; Walker et al., 2002). TdT catalyzes the addition of deoxyribonucleotides to the 30-OH end of DNA strands without any template or primer. This contrasts with most other enzymes that incorporate nucleotides into duplex DNA, as the others generally require a string ofnucleotides on the opposite strand, providing a template that directs the type of nucleotide to be added. Other enzymes require a mixture of all four nucleotides, but TdT can create a homopolymer containing only one type of nucleotide. Typically, deox-yuridine triphosphate (dUTP) is used. TdT labels a variety of 30-OH DNA ends, including double-stranded ends that are blunt, double strands that are protruding or recessed with a 30-hydroxyl single-stranded end, and singlestrand nicks. The DNA end-labeling technique that utilizes TdT is called TdT-mediated dUTP-biotin nick end labeling (TUNEL). We will now describe the modification of the TUNEL method used to detect DNA breaks associated with high NaCl in the mouse renal inner medulla (Dmitrieva et al., 2004), in Caenorhabditis elegans (Dmitrieva et al., 2005), and in marine invertebrates (Dmitrieva et al., 2006).

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