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Molecular and Cellular Biology, December 1999, p. 8353-8360, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Multiple Pathways for Repair of DNA Double-Strand
Breaks in Mammalian Chromosomes
Yunfu
Lin,
Tamas
Lukacsovich, and
Alan S.
Waldman*
Department of Biological Sciences, University
of South Carolina, Columbia, South Carolina 29208
Received 30 April 1999/Returned for modification 10 June
1999/Accepted 9 September 1999
To study repair of DNA double-strand breaks (DSBs) in mammalian
chromosomes, we designed DNA substrates containing a thymidine kinase
(TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a
second defective TK gene sequence to serve as a genetic donor in
recombinational repair. A genomic DSB was induced by introducing
endonuclease I-SceI into cells containing a stably
integrated DNA substrate. DSB repair was monitored by selection for
TK-positive segregants. We observed that intrachromosomal DSB repair is
accomplished with nearly equal efficiencies in either the presence or
absence of a homologous donor sequence. DSB repair is achieved by
nonhomologous end-joining or homologous recombination, but rarely by
nonconservative single-strand annealing. Repair of a chromosomal DSB by
homologous recombination occurs mainly by gene conversion and appears
to require a donor sequence greater than a few hundred base pairs in
length. Nonhomologous end-joining events typically involve loss of very
few nucleotides, and some events are associated with gene amplification
at the repaired locus. Additional studies revealed that precise
religation of DNA ends with no other concomitant sequence alteration is
a viable mode for repair of DSBs in a mammalian genome.
*
Corresponding author. Mailing address: Dept. of
Biological Sciences, University of South Carolina, 700 Sumter St.,
Columbia, SC 29208. Phone: (803) 777-8405. Fax: (803) 777-4002. E-mail: awaldman{at}sc.edu.
Molecular and Cellular Biology, December 1999, p. 8353-8360, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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