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

Nucleic Acids Res. 1998 Apr 1;26(7):1749-54. doi: 10.1093/nar/26.7.1749.

Abstract

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 (rec A-) 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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • 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
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Genotype
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism
  • Recombination, Genetic / genetics
  • Substrate Specificity

Substances

  • DNA, Bacterial
  • DNA, Single-Stranded
  • Oligodeoxyribonucleotides
  • DNA modification methylase PvuII
  • DNA-Cytosine Methylases
  • DNA-Directed DNA Polymerase
  • DNA Restriction Enzymes
  • endodeoxyribonuclease PaeI
  • Deoxyribonucleases, Type II Site-Specific
  • GACGTC-specific type II deoxyribonucleases