Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli.

We studied the effects of double-strand breaks on intramolecular extrachromosomal homologous recombination in mammalian cells. Pairs of defective herpes thymidine kinase (tk) sequences were introduced into mouse Ltk- cells on a DNA molecule that also contained a neo gene under control of the SV40 early promoter/enhancer. With the majority of the constructs used, gene conversions or double crossovers, but not single crossovers, were recoverable. DNA was linearized with various restriction enzymes prior to transfection. Recombination events producing a functional tk gene were monitored by selecting for tk-positive colonies. For double-strand breaks placed outside of the region of homology, maximal recombination frequencies were measured when a break placed the two tk sequences downstream from the SV40 early promoter/enhancer. We observed no relationship between recombination frequency and either the distance between a break and the tk sequences or the distance between the tk sequences. The quantitative effects of the breaks appeared to depend on the degree of homology between the tk sequences. We also observed that inverted repeats recombined as efficiently as direct repeats. The data indicated that the breaks influenced recombination indirectly, perhaps by affecting the binding of a factor(s) to the SV40 promoter region which in turn stimulated or inhibited recombination of the tk sequences. Taken together, we believe that our results provide strong evidence for the existence of a pathway for extrachromosomal homologous recombination in mammalian cells that is distinct from single-strand annealing. We discuss the possibility that intrachromosomal and extrachromosomal recombination have mechanisms in common.

Section: Discussionmentioning

confidence: 99%

An examination of the effects of double-strand breaks on extrachromosomal recombination in mammalian cells.

Yang

Waldman

1992

“…r e d function is not essential for conservative (twoprogeny) double-strand break repair: recA mutations affect the mode and efficiency of homologous recombination stimulated by double-strand breaks in Red and RecE-mediated recombination in several systems (THA-LER et al. 1987;STAHL et al 1974;STAHL and STAHL 1976;NUSSBAUM et al 1992). There has been a proposal that recA function is necessary for consmative or t w e progeny (in our definition) recombination (THALER and STAHL 1988).…”

Section: Intramolecular Double-strand Break Repair I N the A Red Pathwaymentioning

confidence: 97%

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

Yokochi¹,

Kusano²,

Kobayashi³

1995

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.

“…Recombination by the RecE, RecF and X Red pathways is stimulated by double-strand breaks (DSB),' inflicted in vitro or in vivo by restriction endonucleases, or by X terminase activity at X cos sites (SYMING-TON, MORRISON and KOLODNER 1985;STAHL, KOBA-YASHI and STAHL 1985;THALER, STAHL and STAHL 1987a;LUISI-DELUCA, LOVETT and KOLODNER 1989;NUSSBAUM, SHALIT and COHEN 1992). This observation, the high concentration of crossover events near the break (STAHL et al 1974;THALER, STAHL and STAHL 1987b), the inhibitory effect of proteins that protect DNA double-stranded ends (THALER, STAHL and STAHL 1987c) and substrate specificity of enzymes that are involved in these pathways (RADDING 1966;JOSEPH and KOLODNER 1983;LOVETT and KOLODNER 1989) suggest a direct role for DNA ends in the homologous pairing reaction.…”

mentioning

confidence: 99%

“…T h e homologous pairing mechanism may be dictated by substrate configuration and the location of the break with respect to the homologous sequences. A break within one of the two recombining sequences stimulates a RecE-mediated recombination that follows the rules of the DSB-Repair model (KOBAYASHI and TAKAHASHI 1988;NUSSBAUM, SHALIT and COHEN 1992) as put forth for recombination in yeast (RESNICK 1976;ORR-WEAVER and SZOSTAK 1983). In linear intramolecular recombination substrates with direct terminal repeats, the break is located between the homologous sequences (see Figure 1).…”

mentioning

confidence: 99%

“…To gain insight into the nature of the homologous pairing reaction in RecE-mediated intramolecular recombination, by linear substrates with direct terminal repeats, we determined the polarity of the strands incorporated into heteroduplex products. To isolate primary products of intramolecular recombination, we employed a X-vector-based substrate delivery system that facilitates restriction-mediated release of linear substrates within E. coli cells (NUSSBAUM, SHALIT and COHEN 1992). We report here the formation of heteroduplexes by RecE-mediated recombination with a strong bias for paired strands ending 3' at the break.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Heteroduplex strand-specificity in restriction-stimulated recombination by the RecE pathway of Escherichia coli.

Silberstein¹,

Shalit²,

Cohen³

1993

Self Cite

The RecE recombination pathway is active in Escherichia coli recB recC sbcA mutants. To isolate and characterize products and intermediates of RecE-mediated, break-induced, intramolecular recombination, we infected recB recC sbcA mutants, expressing EcoRI endonuclease, with chimeric lambda phages that allow EcoRI-mediated release of cloned linear recombination substrates. Substrates with direct terminal repeats recombined to yield a circular product with one copy of the repeated sequence. Some recombinants were heteroallelic for the recombining markers. Markers distant to the break were recovered in the circular product at a higher frequency than markers close to the break. To examine the heteroduplex structures that may have yielded the heteroallelic recombinants, nonreplicative substrates were employed. Some of the nonreplicative recombination products contained heteroduplexes, with a strong bias for paired strands ending 3' at the break. This strand bias in heteroduplex formation is consistent with recombination models that postulate homologous pairing of protruding 3' single-stranded ends.