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Genetics, Vol 143, 1057-1068, Copyright © 1996
INVESTIGATIONS |
Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage {lambda}-td Intron Model System
M. M. Parker, D. A. Court, K. Preiter and M. Belfort
Molecular Genetics Program, Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, New York 12201-2002
Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage {lambda} model system was developed to analyze exon homology effects on intron homing and determine the role of the {lambda} 5'-3' exonuclease complex (Red{alpha}{beta}) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type {lambda}, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology.
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