47] Specific labeling of newly replicated DNA

Abstract

This chapter discusses the specific labeling of newly replicated DNA. Methods for specific labeling of newly replicated DNA are based on characteristics that distinguish replication of DNA from repair of damaged DNA. DNA replication is a semiconservative process in which one DNA strand acts as the template for the newly synthesized DNA strand. Most genomes are replicated by the replication fork mechanism in which DNA synthesis occurs concomitantly on both templates. DNA synthesis in the direction of fork movement occurs by continuous incorporation of deoxyribonucleotide precursors to form long nascent DNA strands, whereas synthesis in the direction opposite fork movement is carried out discontinuously by the repeated synthesis of short nascent DNA chains referred to as Okazaki fragments. Okazaki fragments provide a useful handle for distinguishing newly replicated DNA from newly repaired DNA. The simplest approach to labeling newly replicated DNA is to label newly synthesized DNA by incorporation of labeled dNTPs at the 3' end of growing DNA chains, and then to characterize the DNA products in order to distinguish DNA replication from DNA repair. Radioactively labeled substrates provide the most sensitive means for detecting nascent DNA. In subcellular systems, any one of the four dNTP substrates for DNA synthesis can be used to label nascent DNA, although addition of two noncomplementary [α- 32 P]dNTPs produces uniformly labeled DNA, regardless of sequence composition. Metabolic redistribution of these radioisotopes to other molecules is not a problem because DNA polymerases incorporate dNTPs directly and rapidly into DNA.