BRIDGING THE GAP - JOINING OF NONHOMOLOGOUS ENDS BY DNA-POLYMERASES

DNA double strand breaks with noncomplementary ends can be joined by m echanisms of nonhomologous recombination. In some systems a DNA end wi th 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 b lunt end with preservation of the 3'-PSS. It has been proposed that th is process occurs via single strand ligation or is facilitated by an a lignment protein. We were interested in testing the hypothesis that a DNA polymerase could function as this putative alignment protein. To c haracterize polymerase activities in this type of reaction, we incubat ed short double-stranded oligonucleotides that had an excess of one of the strands with an exonuclease-free Klenow fragment of Escherichia c oli polymerase I, Tag DNA polymerase from Thermus aquaticus, or an exo nuclease-free Stoffel fragment of Tag DNA polymerase. Products were an alyzed by using biotinylated oligonucleotides separated by denaturing polyacrylamide gel electrophoresis. To further assess the effect of DN A polymerases on the joining of 3'-PSS ends to blunt ends, we incubate d linear plasmid DNA with the polymerases and subjected the DNA to Sou thern blot and sequence analysis. We determined that these DNA polymer ases can use a S'-PSS end as a template after priming off the S'-hydro xyl of a blunt end. This implies that the joining of noncomplementary ends in eukaryotic cells could proceed by a similar mechanism.