Proc. Nati. Acad. Sci. USA Vol. 91, pp. 8527-8531, August 1994 Genetics Homology-associated nonhomologous recombination in mammalian gene targeting (homologous recombln ao/ tIe rembt /gene n ts/genome evolut/p v ) KEIKO SAKAGAMI*, YASUYUKI TOKINAGA*, HIROSHI YOSHIKURAt, AND ICHIZO KOBAYASHI* *Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Shiroganedai, Tokyo 108, Japan; and tDepartment of Bacteriology, Medical School, University of Tokyo, Tokyo 113, Japan Communicated by Susumu Ohno, May 19, 1994 ABSTRACT Nonhomologous (illegitimate) recombinatlon and I.K., unpublished). Preliminary reports of these findings of DNA underlies many changes In the genome. It Involves no have appeared (7, 8). or little homology between recombining DNAs and has been conidered unrelated with homologous recombination, which requres long homology. In mouse cells, however, we found MATERIALS AND METHODS recombination products whose sequences suggest that homol- Plasmds, Bacteria, and Cells. The donor plasmid (pIK39), ogous Interaction between DNAs caused nondologous re- the target plasmid (pIK32) (Fig. 1), and a neo+ version of combination with another DNA. The intermediates of homol- pIK32 (pIK30) have been described (9). Three recAl strains ogous recombination wereapparently trapped at various stages ofE. coli K-12, DH1, DH5, and DH1OB, usedforpropagation and shunted to nonhomologous recombination. In one product, of plasmids, have been described (9, 10) as was a mouse cell the nonhomologous recombination disrupted gene conversion. line, C127 (3). In another, it took place exactly at the end of long homology Establishment of the Target in the Cells. Target plasmid shared between two DNAs. This finding eplains why gene pIK32 was transferred to C127 by calcium phosphate trans- targeting needs long uninterrpted homology and why mam- fection (3). The cells in a focus were recovered with a mailan homologous recombination Is often nonconservative. penicillin cup and spread. Six colonies were subcloned. We discuss possible consequences and roles of this type of Extrachromosomal DNA from each ofthem was isolated by homology-driven gene destruction nism. the Hirt method and was analyzed by the Southern method. One clone carrying plasmid indistinguishable from pIK30was chosen as the target cell line. Its copy number was estimated Several types of DNA recombination have played important to be 160 per cell by a dot blot method with total DNA roles in genome changes and evolution. These include ho- preparation. mologous recombination, which requires long homology be- Gene Targtig to lid DNA. The cell line (C127 car- tween recombining DNAs, and nonhomologous recombina- rying pIK32) was spread to a cell density of 3-4 x 105 cells tion, which requires no or very short homology. These two per 6-cm (diameter) dish. The donor plasmid (10 jg) was recombination mechanisms have been considered quite sep- transferred by a calcium phosphate method (day 0). In arate. experiment 1, 200 jug ofG418 per ml was added on day 3. Its DNA transfer into cultured mammalian cells provides an concentration was increased to 400 jag/ml on day 6. G418R interesting experimental system for the analysis of recombi- (G418-resistant) colonies were counted and isolated with a nation (1). The major route of homologous recombination penicillin cup on day 13. G418 selection was continued until between transferred DNAs is nonconservative (two parental extrachromosomal DNA was prepared by the Hirt method 2 DNAs producing only one progeny DNA) rather than con- months after transfection. In experiment 2, G418 selection servative (two parental DNAs producing two progeny DNAs) started on day 3 at 200 pg/ml. Colony isolation was on day (2, 3). Their nonhomologous recombination is frequent. Ho- 26. DNA was isolated 3 months after transfection. Circular mologous recombination with chromosomal DNA is much DNA plasmids were recovered in E. coli DH1OB by electro- rarer than nonhomologous recombination with it. The fre- poration (Bio-Rad Gene Pulser; 2.5 kV, 25 uF, and 200 fi) by quency of homologous recombination is very dependent on selection for kanamycin resistance. the length of homology and sensitive to minor sequence Sequence Determination and Analysis. Parts ofthe products divergence (4, 5). These properties have been problems in were subcloned in pUC118 and/or pUC119 as described in gene targeting. the figure legends. (For the "donor correction"-type prod- For the analysis of recombination, we have employed a uct, 7881-8246 and 8246-9169 in the coordinate ofthe donor mammalian plasmid shuttle vector derived from bovine pap- were subcloned in pUC118.) DNA sequences were deter- illomavirus type 1 (BPV-1), which replicates in a chromatin- mined with T7 DNA polymerase using the M13 universal like structure in the nuclei of cultured mouse cells (3, 6). We primer (5'-CGACGTTGTAAAACGACGGCCAGT) or the found novel recombination products carrying a nonhomolo- M13 reverse primer (5'-CAGGAAACAGCTATGAC) in a gous joint as well as a homologous joint. Their sequences Pharmacia sequencer. strongly suggest that the process of homologous interaction itself led to the nonhomologous rearrangements. They pro- vide clues to the mechanism of homologous interaction and RESULTS gene targeting in mammalian cells. We detected similar Experimental Design: Gene Targeting to a homology-associated nonhomologous recombination in some Plasmid. Fig. 1A illustrates our recombination substrates. Escherichia coli mutants (K. Kusano, K.S., Y.T., E. Ueda, The target mammalian plasmid consists of BPV-1, an E. coli plasmid and a neomycin phosphotransferase gene (neo) with The publication costs ofthis article were defrayed in part by page charge a deletion. In the first step, we established this target as a payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: BPV, bovine papillomavirus; G418R, G418-resistant. 8527 Downloaded by guest on October 1, 2021 8528 Genetics: Sakagarni et al. Proc. Natl. Acad. Sci. USA 91 (1994) A Substrates B Target correction C Aberrant product '6,z in Hp NT RI RI Donor plasmid 8979 8696 8192 a4 N e~~~~~N q Sal bI 1-,8185 R 1005 7958 1079bp A1 505 A Hr ?889Sa Nr Nr 4433 Bam 4185 Sa Target plasmid _ part of pSV2neo C end deletion _ BP'V- mammalian olasmid neo gene L[] N end deletion S pML2d ( bacterial plasmid ) RI Hp FIG. 1. Experimental system. (A) Substrates. Homologous regions between the donor and the target are drawn as parallel lines. The direction ofthe arrows indicates the direction ofmajor transcription. Restriction enzyme sites are as follows: Bam, BamHI; RI, &oRI; RV, EcoRV; Hin, HindIII; Hp, Hpa I; Sal, SalI; X, Xho I; Na, Nae I; Nr, Nar I. (B) Precise target correction by homologous recombination. The solid line includes joints by homologous recombination. The sequences of the region between deletion a and deletion b and of the 1079-bp long region to the left ofdeletion a in B were shown to be retained. (C) Product with nonhomologous recombination at the end ofhomology. There is gene conversion between the donor and the target at deletion a. Around deletion b, where homology between the donor neo segment (II) and the target neo segment (I) is disrupted, there is nonhomologous recombination (dotted line) between the donor neo DNA (II) and a site outside of the neo homology (III). There is another homologous recombination event outside of the neo homology. Thus the solid line includes two homologous recombination events. From experiment 2. Ps, Pst I site. plasmid in a mouse cell line, taking advantage of the mor- region (II) and the third region (III) took place where the phological transformation. In the second step, we introduced homology between the first region (I) and the second region the donor plasmid, with a neo gene with a different, nonover- (II) ends. This product carried a nonhomologous joint near lapping deletion, into this cell line. Homologous recombina- deletion b, where the homology between the two interacting tion between the two mutant neo genes should restore a neo DNAs (I and II) is interrupted (Fig. 1C). The two functional neo gene and should make the cell resistant to drug homologous DNAs (I and II) experienced a gene conversion G418. The donor plasmid introduced to the cells harboring event, at deletion a, without flanking crossing-over. The the target plasmid produced G418R colonies at a frequency nonhomologousjoint lay only 3 bp away from the point where "5% of that obtained with its neo+ version (Table 1). The the homology ends (I and II) (Fig. 2A). The nonhomologous production ofrecombinant G418R colonies required the pres- joint itselfwas typical in that it involved only 4-bp homology. ence of both target DNA and donor DNA (Table 1). In the Two more independent transformants had exactly the same third step, we recovered these neo+ plasmid molecules in a sequence. This strongly suggests that the nonhomologous recA strain of E. coli by kanamycin selection. joint was formed in the mouse cells rather than in E. coli. The structure of the recovered plasmids was analyzed by This specific configuration immediately suggests its own restriction mapping and sequencing. One species showed origin. The end of the homology-dependent interaction be- precise correction of the mutation of the target by homolo- tween the two DNA segments (I and II) produced some gous recombination (Fig. 1B). A second neo+ species showed unusual structure, like a single-stranded region or a strand precise correction of the donor mutation (data not shown). break, which caused the nonhomologous recombination with We found, however, that neo+ products carrying at least one a third DNA (Ill) (Fig. 2B). The homology-dependent inter- nonhomologous joint constituted the majority (Table 2). action could be strand exchange (Fig. 2C) or protein- Nonhomologous Recombination at the End of Long Homol- mediated alignment and/or protection. ogy. The sequence of the first type of this group strongly Partial Gene Conversion.