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.
Highlights
Illegitimate recombination, in which two ends are joined, typically resulting in a change of sequence
We investigated the DNA polymerase-mediated joining of single- and double-stranded oligonucleotides and constructed a model based on our findings in which both fill-in and overlap products are mediated by DNA synthesis
In Vitro End Joining Reactions—To assess the ability of DNA polymerases to produce overlap products, we set up reactions using discontinuous oligonucleotides as substrate
Summary
Illegitimate recombination, in which two ends are joined, typically resulting in a change of sequence (reviewed for mammalian cells in Ref. 6). Overlap products have been observed in human cells [23], Chinese hamster ovary cells [24], monkey cells [16, 22], Xenopus cell extracts [20, 21], Saccharomyces cerevisiae [25, 26], Schizosaccharomyces pombe [27], and E. coli [28] These overlap products do not have extensive homology, they typically have more complementary bases at the junction site than would be expected for random joining, supporting the assertion that they are not produced by a blunt-ended or single-stranded ligation process. The model is based on simple in vitro experiments and has yet to be demonstrated in vivo
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