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 blunt-end 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.
Mason
R M
RM
DNA Repair and Mutagenesis Group, MRC Radiation and Genome Stability Unit, Harwell, Didcot, Oxon, UK.
Thacker
J
J
Fairman
M P
MP
D000818:Animals; D002417:Cattle; D002457:Cell Extracts / Q000494:pharmacology*; D004249:DNA Damage; D004260:DNA Repair; D018076:DNA, Complementary
D002457:Cell Extracts; D018076:DNA, Complementary