Evidence for conservative (two-progeny) DNA double-strand break repair.

Abstract

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.

Genetic Recombination Through Double-Strand Break Repair: Shift From Two-Progeny Mode to One-Progeny Mode by Heterologous Inserts

“…Inhibition of conservative (two-progeny) doublestrand break repair by a heterologous insert at the break: Figure 2 illustrates the experimental design used to demonstrate the presence of a conservative (two-progeny) double-strand break repair mechanism in an E. coli strain (JC8679) expressing the RecE pathway of homologous recombination (genotype recBC sbcA23) (KOBAYASHI and TAKAHASHI 1988;YOKOCHI et al 1995). Its sbcA23 mutation allows expression of recE and recT genes that are necessary for the double-strand break repair (TAKAHASHI et al.…”

Orientation Dependence in Homologous Recombination

“…198813). Conservative recombination (two recombinant duplex DNA are generated from two parental duplex DNA) by double-strand break repair has been demonstrated (KOBAYASHI and TAKAHASHI 1988;YOKOCHI et al 1995).…”