Nucleic Acids Research, Vol 24, Issue 24 4946-4953, Copyright © 1996 by Oxford University Press
RM Mason, J Thacker and MP Fairman
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.
ARTICLES
The joining of non-complementary DNA double-strand breaks by mammalian extracts
DNA Repair and Mutagenesis Group, MRC Radiation and Genome Stability Unit, Harwell, Didcot, Oxon, UK.
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