pSV2 neo DR SSX. pLCX 102. FIG. 1. (A) Structure of the pSV2neo plasmid. (B) Structure of different pSV2neo derivatives. See Table 6, footnote a for explana-.
Vol. 5, No. 12
MOLECULAR AND CELLULAR BIOLOGY, Dec. 1985, p. 3331-3336 0270-7306/85/123331-06$02.00/0 Copyright © 1985, American Society for Microbiology
Effect of Double-Strand Breaks on Homologous Recombination in Mammalian Cells and Extracts RAJU KUCHERLAPATI* Center for Genetics, College of Medicine, University of Illinois, Chicago, Illinois 60612
KYU-YOUNG SONG, LAVANYA CHEKURI, SIKHA RAUTH, STACY EHRLICH,
AND
Received 29 April 1985/Accepted 29 August 1985
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.
The cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Mouse LMTKcells were also grown in the same medium. Chinese hamster ovary cells (CHO) were grown in Dulbecco modified Eagle medium supplemented with 5% fetal calf serum. 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 changes of the restriction sites did not alter the basic properties of the plasmid. Plasmids that contained polymorphic flanking markers were constructed in the following manner. pLCK 101 was derived from DL by converting the unique EcoRI site to a KpnI site. pLCX 102 was obtained by adding a XhoI site immediately next to the unique BamHI site in pSV2neo DR SSalX. The structures of the various plasmids are shown in Fig. 1. Transfections. Transfection of mammalian cells was achieved by the calcium phosphate coprecipitation method (10). Human cells were usually transfected with 1 ,ug of each parental plasmid per 60 mm petri dish. Transfections were conducted in the absence of carrier DNA. At 4 h, the precipitate was removed, and the cells were treated with 20% dimethyl sulfoxide for 2 min. The cells were transferred to 400 ,ug of G418 per ml on the following day, and resistant colonies were scored after 14 days. Cell extracts. Nuclear extracts were prepared as described by Kucherlapati et al. (8) with the following modifications. The nuclear fraction pellet which was obtained by sonication and centrifugation of the nuclei was suspended in a buffer containing 50 mM Tris (pH 7.5), 10 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 500 mM NaCl,
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, 19-21). 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) have shown that recombination between a yeast chromosomal gene and its homologous counterpart introduced by DNA transfer methods can be enhanced by double-strand cuts in the input plasmid. Based on these and other observations, Szostak et al. (18) proposed a model for homologous recombination, a central feature of which requires a double-strand DNA break to initiate recombination. By using the in vivo and in vitro systems we developed, we now show that double-strand breaks indeed enhance homologous recombination. In addition, the evidence we obtained is consistent with the view that the sites of the double-strand breaks may also be the initiation sites for the recombination event. MATERIALS AND METHODS Cells. Human EJ cells were a gift from R. Weinberg (Massachusetts Institute of Technology, Cambridge, Mass.). *
Corresponding author. 3331
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FIG. 1. (A) Structure of the pSV2neo plasmid. (B) Structure of different pSV2neo derivatives. See Table 6, footnote a for explana-
tion of symbols.
and 10% glycerol (buffer B). This solution was passaged through 3 to 4 ml of DEAE-Sepharose which was extensively washed with buffer B. Protein-rich fractions were identified by absorption at 260 nm, pooled, and precipitated with (NH4)2SO4 (0.313 g/ml). The precipitate was collected by centrifugation, suspended in 1.5 ml of 50 mM Tris-1 mM EDTA-1 mM dithiothreitol-0.1 mM phenylmethylsulfonyl
fluoride-10% glycerol (buffer C), and dialyzed against buffer C. The dialyzed extract was divided and frozen quickly for storage. Nuclear extracts were prepared from human EJ cells, mouse L cells, and CHO cells. DNA manipulations. The conditions of incubation of DNA with cell extracts were described previously (8). After incubating the DNA with the nuclear extracts, the DNA was purified by phenol extractions and ethanol precipitation. A fraction of the purified DNA (usually
