The joining of non-complementary DNA double-strand breaks by mammalian extracts

We have developed a high efficiency system in which mammalian extracts join DNA double-strand breaks with non-complementary termini, This system has been used to obtain a large number of junction sequences from a range of different break-end combinations, allowing the elucidation of the joining mechanisms. Using an extract of calf thymus it was found that the major mechanism of joining was by blunt-end ligation following removal or fill-in of the single-stranded bases. However, some break-end combinations were joined through an efficient mechanism using short repeat sequences and we have succeeded in separating this mechanism from bluntend joining by the biochemical fractionation of extracts, Characterization of activities and sequence data in an extensively purified fraction that will join ends by the repeat mechanism led to a model where joining is initiated by 3' strand invasion followed by pairing to short repeat sequences close to the break site. Thus the joining of double-strand breaks by mammalian extracts is achieved by several mechanisms and this system will allow the purification of the factors involved in each by the judicial choice of the non-complementary ends used in the assay.