Worm Breeder's Gazette 14(3): 23 (June 1, 1996)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Double strand DNA repair in C. elegans

Henri G.A.M. van Luenen1, Philip S. Hartman2, Ronald H.A. Plasterk1

1 The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
2 Texas Christian University, Department of Biology, Box 32916, Forth Worth, TX 76129, United States of America

Transposition of Tc1 and Tc3 is initiated by binding of the respective
transposases to the terminal inverted repeats of each transposon (1,2).
Subsequently, the element is excised by double strand breaks near each
transposon end (3). The cut at the 3' end of the element is between the
last nucleotide of the transposon and the flanking genomic sequence. The
cut at the 5' end is not at the end of the transposon, but two
nucleotides within the transposon. After excision the donor site
contains a double strand break with a two nucleotide extension at each
3' end. The cellular DNA repair processes have to seal the break to
ensure cell viability.

We have set up an in vivo assay to monitor the repair of excision
induced double strand breaks in real time. A Tc3 allele of unc-22
(r750::Tc3) was crossed into a strain containing an inducible Tc3
transposase gene. Upon heat shock induction the transposase gene is
expressed, resulting in transposition of Tc3. Digested genomic DNA of
this strain was analysed on a Southern blot using a fragment of the
unc-22 gene flanking the donor site as a probe. After a two hour
induction at an elevated temperature and a subsequent recovery of the
worms at normal temperature a band appears of the size of the band
expected from the wild type unc-22 gene. This fragment was cloned and
sequenced. It contains the unc-22 sequence with characteristic Tc3
footprints (3). Twenty hours after the induction of transposase
expression approximately 10 % of the unc-22(r750::Tc3) alleles have
reverted. Hybridisation of the blot with the unc-22 probe lead to the
detection of an additional band.  Based on the size and the specific
hybridization of the band we conclude that this band corresponds to the
broken unc-22 fragment created during excision of Tc3. Approximately two
hours after induction this band represent 1-2 % of all unc-22::Tc3
sites. During the remainder of the experiment (up to twenty hours after
induction) the intensity of this band stays constant, whereas the
intensity of the band representing the repaired chromosome continues to
increase. This indicates that the Tc3 elements are continuously excised
and that the broken chromosome is continuously repaired.

The structure of the left end and the right end of the broken chromosome
was determined. The 5' ends and the 3' ends are in agreement with the
structure predicted from the excised transposon (the 3' ends on both
sides of the double strand break contain the two nucleotides which are
not co-excised with the transposon, the 5' ends contain the target
sequence and no transposon sequence).

Thusfar, 3 mutations have been isolated which lead to an increased
sensitivity to DNA damage by radiation (rad-1 (mn155), rad-2 (mn156) and
rad-3 (mn157))(4). Since these mutations might affect DNA break repair,
we tested whether they affect the double strand break repair process
described above. However, none of the mutations had an effect on the Tc3
excision induced double strand breaks.

We have described the first animal model in which the repair of a
genomic double strand DNA break can be monitored in time. It opens the
possibility to study the reaction kinetics and the genes involved in
this repair pathway.

1. Vos, J. C., Van Luenen, H. G. A. M. & Plasterk, R. H. A. (1993) Genes
& Dev. 7, 1244-1253.

2. Colloms, S. D., Van Luenen, H. G. A. M. & Plasterk, R. H. A. (1994)
Nucleic Acids Res. 22, 5548-5554.

3. Van Luenen, H. G. A. M., Colloms, S. D. & Plasterk, R. H. A. (1994)
Cell 79, 293-301.

4. Hartman,P.S., Hevelone, J., Dwarakanath, V. & Mitchell, D.L. (1989)
Genetics 122, 379-385.