Evidence for conservative (two-progeny) DNA double-strand break repair.
The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage lambda Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.
Yokochi
T
T
Department of Molecular Biology, University of Tokyo, Japan.
Kusano
K
K
Kobayashi
I
I
D010582:Bacteriophage lambda / Q000235:genetics; D007446:Chromosome Inversion; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000235:genetics; D005819:Genetic Markers; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D012150:Polymorphism, Restriction Fragment Length; D011938:Rec A Recombinases / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D018741:Replication Origin / Q000235:genetics
D029968:Escherichia coli Proteins; D005819:Genetic Markers; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; C038225:recE protein, E coli