DNA end joining by the klenow fragment of DNA polymerase I

Abstract

DNA end joining is a type of illegitimate recombination characterized by the joining of two DNA ends that lack homology. Using oligonucleotides as substrate, we found that an exonuclease-free derivative of the Klenow fragment of Escherichia coli DNA polymerase I can mediate DNA end joining in vitro. DNA sequence analysis of product DNA indicated that overlap products were formed between direct repeat sequences at the termini of the oligonucleotides. Formation of recombinant products was dependent on the strandedness of the substrate DNA, and the rate of product formation was dependent on the size of the potential overlap. With one to three complementary bases available for pairing at the 3' termini, there was an absolute requirement that one of the oligonucleotides be double-stranded, whereas with four complementary bases, products were also formed in reactions with single-stranded oligonucleotides. When noncomplementary nucleotides were added to the terminus of one of the oligonucleotides, product formation was delayed but not blocked. These data indicate that a DNA polymerase can mediate DNA double strand break rejoining in the absence of other proteins.