The joining of blunt DNA ends to 3′-protruding single strands in Escherichia coli

• Published in: Nucleic acids research 1998-04, Vol.26 (7), p.1749-1754
• Main Authors: King, Jeff, Fairley, Cecilia, Morgan, William
• Format: Article
• Language: English
• Subjects: Base Sequence; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA Damage; DNA Repair - genetics; DNA Restriction Enzymes - metabolism; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; DNA-Cytosine Methylases - metabolism; DNA-Directed DNA Polymerase - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Genotype; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - metabolism; Recombination, Genetic - genetics; Substrate Specificity
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/26.7.1749

In eukaryotic and prokaryotic organisms DNA double-strand breaks with non-complementary ends can be joined by mechanisms of illegitimate recombination. We examined the joining of 3′-protruding single strand (PSS) ends, which do not have recessed 3′ hydroxyls that can allow for fill-in DNA synthesis, to blunt ends. End-joining was examined by electro-transforming Escherichia coli strains with linearized plasmid DNA, sequencing the resulting junctions, and determining the transformation frequencies. Three different E.coli strains were examined: MC1061, which has no known recombination or DNA repair defects, HB101 (recA-) and SURE (recB− recJ−). No striking differences were found in either the spectrum of products observed or the efficiency of end-joining between these strains. As in vertebrate systems, the majority of the products were overlaps between directly repeated DNA sequences. 3′-PSS are frequently preserved in vertebrate systems, but they were not preserved in our experiments unless the transforming DNA was pretreated with a DNA polymerase.