Labeling DNA Breaks In Situ by Klenow Enzyme

Abstract

The Klenow fragment of DNA polymerase I, first described by Klenow and Henningson in 1970 (1), consisted of a 75 kDa proteolytic fragment of the Kornberg polymerase of Escherichia coli. Klenow is now usually obtained as a recombinant protein expressed from a truncated polA gene (2). To polymerize the addition of nucleotides to the 3′-OH end of DNA, Klenow requires a single stranded DNA template and a DNA or RNA primer with a 3′−hydroxyl terminus. Klenow lacks the 5′–3′ exonuclease activity of DNA polymerase I, but retains both the 3′–5′ exonuclease activity, which degrades single stranded or double stranded DNA from a 3′−OH terminus, and the displacement activity of the intact polymerase (3). Applications using Klenow include: end-filling and end-labeling DNA with a 5′-overhang, labeling single stranded DNA by random priming, chain termination sequencing, second strand cDNA synthesis, second strand synthesis in site directed mutagenesis, and production of single stranded probes by primer extension. Of these applications, end-labeling DNA with a 5′-overhang by Klenow has been developed into an in situ technique for the detection of DNA breaks using incorporation of modified nucleotides that may then be detected and visualized by a variety of direct and indirect methods.