Effect of double-strand breaks on homologous recombination in mammalian cells and extracts.

Abstract

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.Purified DNA can be readily introduced into mammalian cells by microinjection (2) or by DNA transfection methods (5). Substantial evidence obtained by the use of viral and plasmid DNA molecules introduced into mammalian cells indicates that homologous recombination between input molecules occurs at a high frequency (3,4,14,15,(19)(20)(21). Based on these original observations, we developed two related systems which permit a dissection of the recombinational process in mammalian cells. One of these is an in vivo system, and the other is a cell-free system that can catalyze homologous recombination between homologous DNA molecules. The central feature of both systems is the use of a eucaryotic-procaryotic shuttle vector, pSV2neo, that carries a gene, which by virtue of its construction can be expressed equally efficiently in mammalian and bacterial cells (16). By using two nonoverlapping, nonreverting deletion mutants of this plasmid, we have shown that mammalian cells are able to mediate homologous recombination at a high frequency (7) and that purified nuclear extracts from human and rodent cells can mediate homologous recombination between these plasmids (8).Orr-Weaver et al. (13) Plasmids. pSV2neo plasmid was constructed by Southern and Berg (16). The deletion derivatives of pSV2neo were described previously (7). Briefly, pSV2neo DL (DL) was prepared by removing a 248-base-pair (bp) NarI fragment at the 5' end of the gene. pSV2neo DR (DR) was constructed by deletion of a 283-bp NaeI fragment. The unique NaeI site in DR was converted to a Sall site by linker addition (12). This plasmid is referred to as pSV2neo DR S. The deleted neo gene in this plasmid is flanked by a HindIlI site at the 5' end and by a SmaI site at the 3' end. These sites were changed to a SmaI and an XbaI site, respectively. This modified plasmid is referred to as pSV2neo DR SSalX. The chang...

Intermolecular homologous recombination in plants.

“…Although a double-strand break is reported to stimulate recombination in animal cells (1,10,11,38), we observed no such stimulation when providing one plasmid with a doublestrand break. This may point to a mechanism other than the double-strand break repair model proposed by Szostak et al (40).…”

Homologous recombination enhancement conferred by the Z-DNA motif d(TG)30 is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences.

“…There are 501 bp of homology between the sites of the two deletions; recombination events in this interval between DL and DR plasmids can regenerate a wild-type neo gene that can confer G418 resistance in mammalian cells. Derivatives of DL and DR containing multiple restriction site modifications are available (38). Sequences under investigation were cloned between the StuI and HindIII sites of pSV2neo and the two deletion plasmids pLCKS (DLTG) and pDR-D86 (DRTG).…”

Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates.

“…A requirement of the model is that ends of the repaired DNA molecule be located in a region of homology shared by both DNA molecules. While this model can account for the high level of recombination between certain of the DNA substrates introduced into mammalian cells (3,18,36), it does not effectively explain other efficient recombination events generated by introducing into cells DNAs with ends located in nonhomol-* Corresponding author. …”