Bridging the gap. Joining of nonhomologous ends by DNA polymerases.
DNA double strand breaks with noncomplementary ends can be joined by mechanisms of nonhomologous recombination. In some systems a DNA end with a 3'-protruding single strand (PSS), which does not have a recessed 3'-hydroxyl that can allow for fill-in DNA synthesis, is joined to a blunt end with preservation of the 3'-PSS. It has been proposed that this process occurs via single strand ligation or is facilitated by an alignment protein. We were interested in testing the hypothesis that a DNA polymerase could function as this putative alignment protein. To characterize polymerase activities in this type of reaction, we incubated short double-stranded oligonucleotides that had an excess of one of the strands with an exonuclease-free Klenow fragment of Escherichia coli polymerase I, Taq DNA polymerase from Thermus aquaticus, or an exonuclease-free Stoffel fragment of Taq DNA polymerase. Products were analyzed by using biotinylated oligonucleotides separated by denaturing polyacrylamide gel electrophoresis. To further assess the effect of DNA polymerases on the joining of 3'-PSS ends to blunt ends, we incubated linear plasmid DNA with the polymerases and subjected the DNA to Southern blot and sequence analysis. We determined that these DNA polymerases can use a 3'-PSS end as a template after priming off the 3'-hydroxyl of a blunt end. This implies that the joining of noncomplementary ends in eukaryotic cells could proceed by a similar mechanism.
King
J S
JS
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143.
Fairley
C F
CF
Morgan
W F
WF
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013824:Thermus / Q000201:enzymology
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase