Strand Displacement during Deoxyribonucleic Acid Synthesis at Single Strand Breaks

Abstract Synthesis of DNA by DNA polymerases has been studied by using circular duplex DNA templates which contain single phosphodiester bond interruptions (nicks). Escherichia coli DNA polymerase can initiate synthesis at nicks by the covalent attachment of nucleotides to the 3' end of the primer strand. An initial phase of synthesis is accompanied by the removal of nucleotides from the 5' end of the nick by the 5'-hydrolytic activity of the E. coli polymerase (nick translation). After the incorporation of 10 to 50 nucleotides, the 5' end of the primer strand is displaced by the growing 3' end of the primer strand. Elimination of the 5'-hydrolytic activity of the E. coli polymerase does not prevent this strand displacement. In contrast, phage T4 polymerase cannot initiate synthesis at nicks. In these studies polynucleotide ligase has been used to identify nicks and to measure their disappearance during strand displacement. The DNA synthesized during the phase of strand displacement is sensitive to both exonucleases I and III of E. coli. This observation suggests that an equilibrium exists between single and double stranded forms of the newly synthesized DNA. A third phase of more extensive synthesis yields a DNA product which is resistant to exonuclease I even after denaturation, suggesting a self-complementary structure. Electron micrographs of such a product synthesized on circular PM2 DNA containing one nick per molecule reveal a single branch extending from the intact circle. Subsequent synthesis gives rise to multiple branches.