Proc. Nati. Acad. Sci. USA Vol. 91, pp. 8527-8531, August 1994 Homology-associated nonhomologous recombination in mammalian 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 , 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ABSTRACT Nonhomologous (illegitimate) recombinatlon and I.K., unpublished).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 and has been conidered unrelated with , which requres long homology. In cells, however, we found MATERIALS AND METHODS recombination products whose sequences suggest that homol- Plasmds, , 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 . 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 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 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. The product in Fig. 3A, the suggested that homologous interaction caused the nonhomol- second type with a nonhomologous joint, shows another ogous recombination. As diagrammed in Figs. 1C and 2B, the mode of association of homologous and nonhomologous nonhomologous recombination between the second DNA recombination. The sequence of the upper neo segment Table 1. Formation of recombinant colonies G418R G418R colonies/ Recombination Exp. Target Donor* colonies transfected cell efficiency,t % 1 neoAC neoAN 15; 11 3.7 x 10-5 7 neoAC neo+ 188; -200 -5.5 x 10-4 100 neoAC None 0; 0 <1.4 x 10-6 <0.5 2 neoAC neoAN 6; 8 2.0 x 10-5 4 neoAC neo+ "200; "200 -5.7 x 10-4 100 neoAC None 0; 0 <1.4 x 10-6 <0.5 None neoAN 0; 0 <1.4 x 10-6 <0.5 None neo+ 112; 87 2.8 x 10-4 100 None None 0; 0 <1.4 x 10-6 <0.5 neo+ indicates pIK30. *Input was 10 pg per 6-cm (diameter) dish. tRecombination efficiency is defined as [number of G418R colonies]/[number of G418R colonies from the neo+ donor plasmid (pIK30)]. Downloaded by guest on October 1, 2021 Genetics: Sakagami et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8529

Table 2. Classification of the products the plane of paper would overlap it with the other product Homologous (Fig. 4AiR). In one (i), the nonhomologous recombination joint only took place at a site very close to the homologous recombi- nation event that reconstituted neo+ (199 bp to the left of Target Donor Homologous joint and deletion b). In the second (ii), the nonhomologous recombi- Exp. corrected corrected nonhomologous joint Total nationjoint was close to the homologous recombination (184 1* 1 (1) 0 (0) 3 (2) 4 (3) bp to the left of deletion a). In both the products, it appears 2t 0(0) 2 (1) 37 (7) 39(8) as if the nonhomologous recombination joined the end "left The numbers outside the parentheses refer to the number ofE. coli behind" by nonconservative homologous recombination. transformants obtained. The numbers inside the parentheses refer to The nonhomologous recombination involved a very short (1 the number of plasmid types as classified by 6-bp restriction en- bp or 5 bp) sequence homology (Fig. 4B). zymes. Another common structural feature is the presence of *Plasmid preparation from each of three G418R clones was used for inverted repeats at the nonhomologous recombination site transfer to E. coli separately. Four colonies were recovered. Two that can potentially form a stem-loop (upper DNAs in Fig. of the kanamycin-resistant transformants from one clone had the same type of plasmid with an identical nonhomologous joint. 4B). In product i, the potential stem-loop scores 27 points in tPlasmid preparation from a mixture of 6 G418R clones was used for program STEM-LOOP ofthe University ofWisconsin Genetics transfer to E. coli. Finding multiple recombinants within one Computer Group. There is no potential stem-loop more recipient colony is frequent in calcium phosphate transfection of stable than this in a region between this one and the right mammalian cells (for example, ref. 11) because many DNA mole- boundary of deletion a (see Fig. 4Ai), which should include cules are taken up by one cell (12). the site of the neo+ homologous recombination event. In ii, the stem-loop scores 23 points. There is none more stable corresponding to deletion a (283 bp long; from coordinate than this between this and the left boundary ofdeletion a. We 8979 through coordinate 86%) was copied from the left end discuss their possible significance later. We confirmed each for only 155 bp (coordinate 8979 through 8824). There the of these nonhomologous joint sequences with another inde- DNA was joined to an unrelated site (coodinate 6405 of the pendent E. coli transformant. target) by nonhomologous recombination (Fig. 3B). Thejoint We analyzed sequence ofthe fifth type ofproduct showing involved 0-bp homology. association of homologous recombination and nonhomolo- Nonconservative Recombination. The above product (Fig. gous recombination. It can be explained by nonconservative 3) represents nonconservative (one-progeny) homologous homologous recombination between the neo gene in a head- recombination. The two products depicted in Fig. 4 (the third to-tail, circular dimer form of the donor plasmid and the neo type and the fourth type) also belong to this class. They share gene of the target plasmid, followed by joining of the two the same configuration in spite of their different contents. resulting ends after extensive degradation (between a donor Rotation of one product (Fig. 4Ai) 1800 around its center on site between 4505 and 5608 and a target site between 9519 and

C Homology - j0~~3~C~% Homology' -.W t°onbOMo I. I Rl..~- 6 TTGGCCCGCGGGN (Target) AACCGGGCGCCCC I x 8185 XI R II Sal _- - TTGGCCCGCGGGG TCGA>TAAGGi.L RV (Donor) ..AACCGGGCGCCCC A6CTGGCAATTCC- Homologous pairing N 819c77

.-.~ohomologous C4Wco..<,,,recombination Nonhomologous Branch migration recombination with ilL. -' ...v..a third unrelated|ss DNA

CCGCGGGGTCGACCGTCTTCA GGCGCCCCAGCTGGCAGAAGT

B Homology * 1 > Resolution Nonomloou Conservative homologous recombination

m

Nnhomologous recombinaton

FIG. 2. Nonhomologous recombination at the end ofhomology. (A) Sequences. The product in Fig. 1C. There is sequence homology between the donor (II) and the target (I) up to the site one base pair away from the Nar I sites (the dotted bases). The sequence between the Pst I (donor coordinate 8246) site ofthe donor and the HindIII site ofthe target (see Fig. 1C) was determined. (B) Diagram. The middle DNA (II) shares sequence homology with the upper DNA (I) in the left side. Exactly at the end of this homology, the middle DNA (I) experiences nonhomologous recombination with the lower DNA (III). (C) Possible common mechanism. (i) Homologous pairing at the long homology results in a Holliday-type intermediate. Destructive (nonconservative) resolution ofthe nascent Holliday structure leads to the product in Fig. 3. (ii) The Holliday structure moves along the homology by branch migration. Encounter with stem-oop or other unusual structure triggers its destructive resolution. The net result is nonconservative recombination and nonhomologous recombination as for the products in Fig. 4. (iii) When the Holliday structure encounters nonhomology, nonhomologous rearrangements take place to produce the recombinant in Fig. 1C and Fig. 2A and B. Downloaded by guest on October 1, 2021 8530 Genetics: Sakagami et al. Proc. Natl. Acad. Sci. USA 91 (1994)

A B 8860O Partial gene conversion 8824 Donor \ Nae _-- - CTCAGAAGAATAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACG [4(ono (Donor) ...... if 8979I 8696 CTTTACTTGGGTTGTGAACAGTCCTGTCAGGTAGTGCAAATTGATTGGGATCAGGTAGTTGTATTTTAAATACCCTA -- -o-Barn 6405 6369 (Target) Nae _ - -- CTCAGAAGAATAACTCGTCAAGAAGGCGATAGAAGGCGATTTGATTGG&GATCAGGTAGTTGTATTTTAAATACeCCTA -D- Barm

Target Bam Cla 405 FIG. 3. Partial gene conversion. (A) Product. Cla, Cla I site; Hp, Hpa I site. From experiment 2. (B) Sequences. Partial gene conversion copying the donor DNA took place to the left of the nonhomologous recombination site in the upper sequence. The sequence between the Hpa I site (coordinate 2737) and the Cla I site (coordinate 6038) of the target (see A) was determined. 10622) (data not shown). This type of nonconservative re- peats of a BPV vector is frequent in the mouse cells (3). (c) combination has been observed previously in polyomavirus We constructed the circular dimer plasmid that will be DNA (13). produced by reciprocal recombination in step i and intro- Nonhonologous RecoMnbiaon iln Cels Instead duced it into the same E. coli strain. It excised the BPV unit of E. coi Cels. The following strongly argues that the by the predicted homologous recombination (data not nonhomologous joint in these products was formed in the shown). mouse cells rather than in the E. coli cells. First, independent The product with an entire BPV part (Fig. 4Ai) can be E. colitransformants showed exactly the samejoint sequence explained by a similar route starting with homologous re- (first, third, and fourth types). Second, we were able to detect combination between the pML parts. a frgment of the same size as the one carrying the nonho- mologous joint in the original mouse cell extract by PCR (third type) (data not shown). Third, we have not detected DISCUSSION nonhomologous recombination of these substrates in the E. Mechanism. The above results demonstrate that the non- coli strain we have used. homologous joint and the neo+ homologous joint in the Some of the products lacked BPV sequences and will not products were formed in the mouse cells rather than in the E. replicate in the mouse cells for the long period of selection. colf cells. We assume that these two recombination events Our working hypothesis for this is the following multistep were associated. Their sequences are consistent with our reaction. (i) The donor and the target are engaged in homol- assumption, although it is difficult to rigorously exclude the ogous recombination at the BPV part to form a dimer in a possibility that an initial neo+ homologous recombinant mouse cell. (ii) The neo+ homologous recombination and the experienced subsequent nonhomologous recombination nonhomologous recombination take place and produce re- combinants carrying a pair of direct repeats of the pML events during growth ofthe mouse cells. In harmony with our (bacterial plasmid) part. The product replicates during the assumption, we could not identify sequences previously G418 selection. (iii) It excises one unit of BPV part by implicated in nonhomologous rearrangements around the intramolecular homologous recombination between the di- joints, except for the inverted repeats mentioned (data not rect repeats. This homologous recombination takes place shown). either in the mouse cells just before harvest of DNA or in E. One common mechanism, the destruction ofHolliday-type col just after transfer. intermediates (illustrated in Fig. 2C), can explain the gener- The following observations are consistent with this route. ation of these products economically. (i) Two DNAs sharing (a) We detected many plasmid molecules of the size of the homology in one region recognize each other and form a predicted dimeric recombinant and few (-.1/100) plasmid cross-stranded (Holliday) structure. (ii) This intermediate molecules of the size of the recombinant plasmid (Fig. 4Aii) structure moves along the homology, in a process known as in the DNA preparation from the mouse clone (data not branch migration. (iii) When it encounters nonhomology or shown). (b) Homologous recombination between direct re- stem-loop structure, an unusual structure such as a break

A (i) A (ii) B (i) neo8homologous joint < - - Knverted repeats. 8354 CGCC-GCGTRACGTFGTCGCTflCTFCCTiTGfPlflGCC0TCCTC TCCFCTCT PCTTCCTCTSGGCCGC.CCCGT- -*-RV 7 Sal-a---ACFGTTCTTCTPOGP CTPO Donor) Sal --REfcTCCCCC....c-FC C: CTPGC, T f- A^FCX-PcPICccCT TCr N cT 11135 1 Sal l (Donorl Sal A-CT-RE TTCTTCTAGCAFFCTFGr-PCC-ccccCTI4CCTCCTICFCICTRCTCTCCTCTPCCFCPCCG CCT-- -a RV . . .,.,......

34 j B (ii) 3461 inverted repeats - neo+ homologous joint ( RI- - PRP GCGGCCPTCGTGECCTCCCCPCTCCTCCPGTTCGGcOGCETCCeFTGCOCGFTPCCCCCTCCTC7'.TCC- GGFT 'Bam (Target) CCFCPTGTTTCTGCPrTCCFCCCTTGCFCCToCCGPCPCCCCTCGT;pT C.rG:CPC'CPTCePCPCcr'CTfCTTFL - Ball + 5333 52a9 (Target)

RIt ~a-- PF.0 n scX GcCPiTc TCCTCCCCFCc TCCT GeCPc TTCG DCG C TC f-T r;FC Gf;lGAiC-fCTT c;5PPTCGq cA;F'TcF F _ - - Barn *...... e~sX.S FIG. 4. Nonconservative recombination. (A) Products. (i) From experiment 1. (ii) From experiment 2. (B) Sequences. (i) The homologous recombination event generating neo+ took place to the left of the upper sequence shown (see Ai). The inverted repeats (dotted arrows) can potentially form a stem-loop. The sequence between the Sal I site and the EcoRV site ofthe donor (see Ai) was determined. (ii) The homologous recombination event generating neo+ took place to the right of the upper sequence shown (see All). The inverted repeats can potentially form a stem-loop. The sequence between the EcoRI site and the BamHI site of the target (see Aii) was determined. Downloaded by guest on October 1, 2021 Genetics: Sakagami et al. Proc. NatL. Acad. Sci. USA 91 (1994) 8531 forms and causes nonhomologous rearrangement of one of Other Homology-Dependent Gene Inactivation Phenomena. the DNAs with a third DNA. We detected this type of homology-associated nonhomolo- The particular product in Fig. 3 can be explained by gous recombination in some E. coli mutants (K. Kusano, destruction of a nascent Holliday-like structure with accom- K.S., and I.K., unpublished). There are homology-depen- panying nonhomologous recombination. Other possible dent gene inactivation processes involving point mutagenesis mechanisms include the following: (i) DNA synthesis copy- (RIP) (24, 25), methylation (MIP) (26), and heterochromatin- ing the sequence a paused, and the end of newly synthesized ization (27). Head-to-head diner formation could be another DNA was joined to the unrelated site (copy-join). (ii) DNA example (28). synthesis switched templates (copy-choice). (iii) Heterodu- plex of the deletion and its wild-type allele caused the We thank Dr. David Leach, Dr. Frank Stahl, and Dr. Masami Hasegawa for reading earlier versions of this manuscript. Dr. nonhomologous recombination. Susumu Ohno, Dr. Werner Arber, Dr. Maria Jasin, Dr. Mitiko Go, A major route of homologous recombination between Dr. Brooks Low, Dr. Maurice S. Fox, Dr. Jeff Strathern, Dr. Pierre DNA transferred to mammalian somatic cells is nonconser- Chartrand, Dr. Dana Carrol, and Dr. Yoshihiro Kitamura provided vative (one-progeny type). The products in Figs. 3 and 4 encouragement and/or advice. The work at the Institute of Medical suggest one way in which the homologous interaction be- Science was supported by grants to I.K. from Uehara Science tween two intact duplexes can result in only one progeny. Foundation, Nissan Science Foundation, Department of Health The homologous interaction itself, more specifically, the act (), and Department of Education (gene therapy, stress of nonconservative resolution of the intermediates, may response, recombination) and, in part, by grants to Dr. Hideo Ikeda generate one homologous recombinant and some other ab- (Department Head). errant structure such as a break, which causes nonhomolo- 1. Bollag, R. J., Waldman, A. S. & Liskay, R. M. (1989) Annu. gous recombination (Fig. 2C). Consistent with this scheme, Rev. Genet. 23, 199-225. one ofthe two ends of a linear donor DNA can participate in 2. Chakrabarti, S. & Seidman, M. M. (1986) Mol. Cell. Biol. 6, homologous recombination and the other in nonhomologous 2520-2526. recombination (14, 15). A similar succession of end- 3. Kitamura, Y., Yoshikura, H. & Kobayashi, I. (1990) Mol. Gen. generating nonconservative recombination (half crossing- Genet. 222, 185-191. was for E. the second recom- 4. Capecchi, M. R. (1989) Science 244, 1288-1292. over) proposed coli, although 5. te Riele, H., Robanus Maandag, E. & Berns, A. (1992) Proc. bination event is homologous rather than nonhomologous Natl. Acad. Sci. USA 89, 5128-5132. (16, 17). 6. Bertino, A. M., Tischfield, J. A. & Stambrook, P. J. (1992) There are corresponding observations in vitro with two Mol. Gen. Genet. 232, 24-32. purified recombination enzymes. Torsional stress generated 7. Kusano, K., Sakagami, K., Ueda, E. & Kobayashi, I. (1993) by RecA protein during DNA strand exchange separates Cold Spring Harbor Symp. Quant. Biol. 58, 100 (abstr.). strands ofa heterologous insert (18). T4 VII, a 8. Kusano, K., Sakagami, K., Ueda, E. & Kobayashi, I. (1993) J. Holliday junction resolvase, can also cleave the junction Cell. Biochem., Suppl. 17E, 298. between single-stranded and double-stranded DNA in a de- 9. Yamamoto, K., Yoshikura, H., Takahashi, N. & Kobayashi, I. letion heteroduplex (19). (1988) Mol. Gen. Genet. 212, 393-404. 10. Grant, S. G. N., Jessee, J., Bloom, F. R. & Hanahan, D. (1990) The homology-associated nonhomologous recombination Proc. Natl. Acad. Sci. USA 87, 4645-4649. mechanisms- might be responsible for aberrant products in 11. Lin, F.-L., Sperle, K. & Stemnberg, N. (1984) Mol. Cell. Biol. gene targeting (20, 21). 4, 1020-1Q34. Fudrter Implications for Gene Targeting. Mammalian gene 12. Perucho, M., Hanahan, D. & Wigler, M. (1980) Cell 22, targeting frequency is very dependent on the homology 309-317. length up to 10 kb (4) and is sensitive to rare sequence 13. Brouillette, S. & Chartrand, P. (1987) Mol. Cell. Biol. 7, differences scattered over the homology (5). The homology- 2248-2255. associated nonhomologous recombination mechanism can 14. Berinstein, N., Pennell, N., Ottaway, C. A. & Shulman, M. J. provide an answer to this mystery as follows. We hypothe- (1992) Mol. Cell. Biol. 12, 360-367. 15. Hasty, P., Rivera-Perez, J. & Bradley, A. (1992) Mol. Cell. size that Holliday-type intermediates of homologous inter- Biol. 12, 2464-2474. action are easily formed in gene targeting. They move along 16. Takahashi, N. K., Yamamoto, K., Kitamura, Y., Luo, S., DNA as long as there is homology between two DNAs, Yoshikura, H. & Kobayashi, I. (1992) Proc. Natl. Acad. Sci. before successful resolution into homologous recombinants, USA 89, 5912-5916. and are destroyed at the ends of homology (Fig. 2C) (or they 17. Yamamoto, K., Kusano, K., Takahashi, N., Yoshikura, H. & just run off). The longer the homology, the smaller the chance Kobayashi, I. (1992) Mol. Gen. Genet. 234, 1-13. to meet the ends of the homology and, hence, the larger the 18. Jwang, B. & Radding, C. M. (1992) Proc. Nat!. Acad. Sci. USA chance for successful resolution. This formulation explains 89, 7596-7600. quantitatively the length dependence in mammalian gene 19. Kleff, S. & Kemper, B. (1988) EMBO J. 7, 1527-1535. 20. Adair, G. M., Nairn, R. S., Wilson, J. H., Seidman, M. M., targeting (Y. Fujitani, K. Yamamoto, and I.K., unpublished). Brotherman, K. A., Mackinnon, C. & Scheerer, J. B. (1989) Rare, scattered sequence differences would destroy the in- Proc. Natl. Acad. Sci. USA 86, 4574-4578. termediates similarly when the intermediates encounter 21. Aratani, Y., Okazaki, R. & Koyarna, H. (1992) Nucleic Acids them. Res. 20, 4795-4801. Possible Roles and Possible Cotbutlon to Genome Rear- 22. Ohno, S. (1970) Evolution by Gene Duplication (Springer, rangements. We discuss the possibility that this homology- Berlin). associated nonhomologous recombination mechanism under- 23. Kobayashi, I., Adv. Biophys. 31, in press. lies several types of genome rearrangements and gene dupli- 24. Selker, E. U. (1990) Annu. Rev. Genet. 24, 579-613. cation (22) elsewhere (I.K., unpublished). This mechanism 25. Kricker, M. C., Drake, J. W. & Radman, M. (1992) Proc. Natl. Acad. Sci. USA 89, 1075-1079. might have a positive role, such as abortion of erroneous 26. Faugeron, G., Rhounim, L. & Rossignol, J. (1990) Genetics homologous recombinationbetween shortrepeats andgenome 124, 585-591. destruction under a special condition as described elsewhere 27. Henikoff, S. & Dreesen, T. (1989) Proc. Nat!. Acad. Sci. USA (23). We do not know yet whether we can generalize this 86, 6704-6708. inding with this experimental situation on the overall process 28. Kunes, S., Botstein, D. & Fox, M. S. (1985) J. Mo!. Biol. 184, of . 375-387. Downloaded by guest on October 1, 2021