Fine-resolution analysis of products of intrachromosomal homeologous recombination in mammalian cells.

Mouse Ltk- cell lines that contained a herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene with a 16-bp insertion mutation linked to either a defective HSV-2 tk gene or a hybrid tk sequence comprised of HSV-1 and HSV-2 tk sequences were constructed. HSV-1 and HSV-2 tk genes have 81% nucleotide identity and hence are homeologous. Correction of the insertion mutant HSV-1 tk gene via recombination with the hybrid tk sequence required an exchange between homeologous tk sequences, although recombination could initiate within a region of significant sequence identity. Seven cell lines containing linked HSV-1 and HSV-1-HSV-2 hybrid tk sequences gave rise to tk+ segregants at an average rate of 10(-8) events per cell division. DNA sequencing revealed that each recombinant from these lines displayed an apparent gene conversion which involved an accurate transfer of an uninterrupted block of information between homeologous tk sequences. Conversion tract lengths ranged from 35 to >330 bp. In contrast, cell lines containing linked HSV-1 and HSV-2 tk sequences with no significant stretches of sequence identity had an overall rate of homeologous recombination of <10(-9). One such cell line produced homeologous recombinants at a rate of 10(-8). Strikingly, all homeologous recombinants from this latter cell line were due to crossovers between the HSV-1 and HSV-2 tk genes. Our results, which provide the first detailed analysis of homeologous recombination within a mammalian genome, suggest that rearrangements in mammalian genomes are regulated by the degree of sequence divergence located at the site of recombination initiation.     

Suppression of intrachromosomal gene conversion in mammalian cells by small degrees of sequence divergence

Pairs of closely linked defective herpes simplex virus (HSV) thymidine kinase (tk) gene sequences exhibiting various nucleotide heterologies were introduced into the genome of mouse Ltk- cells. Recombination events were recovered by selecting for the correction of a 16-bp insertion mutation in one of the tk sequences. We had previously shown that when two tk sequences shared a region of 232 bp of homology, interruption of the homology by two single nucleotide heterologies placed 19 bp apart reduced recombination nearly 20-fold. We now report that either one of the nucleotide heterologies alone reduces recombination only about 2.5-fold, indicating that the original pair of single nucleotide heterologies acted synergistically to inhibit recombination. We tested a variety of pairs of single nucleotide heterologies and determined that they reduced recombination from 7- to 175-fold. Substrates potentially leading to G-G or C-C mispairs in presumptive heteroduplex DNA (hDNA) intermediates displayed a particularly low rate of recombination. Additional experiments suggested that increased sequence divergence causes a shortening of gene conversion tracts. Collectively, our results suggest that suppression of recombination between diverged sequences is mediated via processing of a mispaired hDNA intermediate.     

Accurate homologous recombination is a prominent double-strand break repair pathway in mammalian chromosomes and is modulated by mismatch repair protein Msh2

We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a “donor” tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.     

An Examination of the Effects of Double-Strand Breaks on Extrachromosomal Recombination in Mammalian Cells

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.     

Requirements for ectopic homologous recombination in mammalian somatic cells.

Ectopic recombination occurs between DNA sequences that are not in equivalent positions on homologous chromosomes and has beneficial as well as potentially deleterious consequences for the eukaryotic genome. In the present study, we have examined ectopic recombination in mammalian somatic (murine hybridoma) cells in which a deletion in the mu gene constant (Cmu) region of the endogenous chromosomal immunoglobulin mu gene is corrected by using as a donor an ectopic wild-type Cmu region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal mu gene deletions of 600 bp and 4 kb are corrected less efficiently than a deletion of only 2 bp, (ii) the minimum amount of homology required to mediate ectopic recombination is between 1.9 and 4.3 kb, (iii) the frequency of ectopic recombination does not depend on donor copy number, and (iv) the frequency of ectopic recombination in hybridoma lines in which the donor and recipient Cmu regions are physically connected to each other on the same chromosome can be as much as 4 orders of magnitude higher than it is for the same sequences located on homologous or nonhomologous chromosomes. The results are discussed in terms of a model for ectopic recombination in mammalian somatic cells in which the scanning mechanism that is used to locate a homologous partner operates preferentially in cis.     

The mechanism of mammalian gene replacement is consistent with the formation of long regions of heteroduplex DNA associated with two crossing-over events

In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin mu-delta region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCmuCdeltapal) consists of pSV2neo sequences bounded on one side by homology to the mu gene constant (Cmu) region and on the other side by homology to the delta gene constant (Cdelta) region. The Cmu and Cdelta flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cmu and Cdelta regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cmu and Cdelta regions. In several recombinants, palindrome markers in the Cmu and Cdelta regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.     

The Search for Homology Does Not Limit the Rate of Extrachromosomal Homologous Recombination in Mammalian Cells

Mouse LTK(-) cells were transfected with a pair of defective Herpes simplex virus thymidine kinase (tk) genes. One tk gene had an 8-bp insertion mutation while the second gene had a 100-bp inversion. Extrachromosomal homologous recombination leading to the reconstruction of a functional tk gene was monitored by selecting for tk positive cells using medium supplemented with hypoxanthine/aminopterin/thymidine. To assess whether the search for homology may be a rate-limiting step of recombination, we asked whether the presence of an excess number of copies of a tk gene possessing both the insertion and inversion mutations could inhibit recombination between the singly mutated tk genes. Effective competitive inhibition would require that homology searching (homologous pairing) occur rapidly and efficiently. We cotransfected plasmid constructs containing the singly mutated genes in the presence or absence of competitor sequences in various combinations of linear or circular forms. We observed effective inhibition by the competitor DNA in six of the seven combinations studied. A lack of inhibition was observed only when the insertion mutant gene was cleaved within the insertion mutation and cotransfected with the two other molecules in circular form. Additional experiments suggested that homologous interactions between two DNA sequences may compete in trans with recombination between two other sequences. We conclude that homology searching is not a rate-limiting step of extrachromosomal recombination in mammalian cells. Additionally, we speculate that a limiting factor is involved in a recombination step following homologous pairing and has a high affinity for DNA termini.     

Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process.

We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity. The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+. Using this system, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells. The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands. This process exposes complementary strands of the two defective tk genes, which then pair. Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.     

Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes

To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.     

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.     

Meiotic recombination in Drosophila Msh6 mutants yields discontinuous gene conversion tracts

Crossovers (COs) generated through meiotic recombination are important for the correct segregation of homologous chromosomes during meiosis. Several models describing the molecular mechanism of meiotic recombination have been proposed. These models differ in the arrangement of heteroduplex DNA (hDNA) in recombination intermediates. Heterologies in hDNA are usually repaired prior to the recovery of recombination products, thereby obscuring information about the arrangement of hDNA. To examine hDNA in meiotic recombination in Drosophila melanogaster, we sought to block hDNA repair by conducting recombination assays in a mutant defective in mismatch repair (MMR). We generated mutations in the MMR gene Msh6 and analyzed recombination between highly polymorphic homologous chromosomes. We found that hDNA often goes unrepaired during meiotic recombination in an Msh6 mutant, leading to high levels of postmeiotic segregation; however, hDNA and gene conversion tracts are frequently discontinuous, with multiple transitions between gene conversion, restoration, and unrepaired hDNA. We suggest that these discontinuities reflect the activity of a short-patch repair system that operates when canonical MMR is defective.     

Analysis of spontaneous gene conversion tracts within and between mammalian chromosomes

In the present study, we report the first characterization of gene conversion tract length, continuity and fidelity for pathways of gene targeting, ectopic and intrachromosomal homologous recombination using the same locus and mammalian somatic cell type. In this isogenic cell system, the vast majority of recombinants (> 97%) are generated by homologous recombination and display a high degree of fidelity in the gene conversion process. Individual gene conversion tracts are highly likely to involve single, independent recombination events and proceed through a heteroduplex DNA intermediate. In all recombination pathways, gene conversion tracts are long, extending up to ∼ 2 kb. Most gene conversion tracts are continuous in favor of donor region sequences, but in a small fraction of recombinants (15%), discontinuous gene conversion tracts are observed. In most cases, the recombination donor sequence is unaltered, although in two cases of intrachromosomal recombination, both recombination donor and recipient sequences bear gene conversion tracts. Overall, gene conversion events are similar, both qualitatively and quantitatively, for homologous recombination within and between mammalian chromosomes.     

Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells

Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93-101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2(-/-) cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.     

Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.

We established a mouse Ltk- cell line that contains within its genome a herpes simplex virus thymidine kinase gene (tk) that had been disrupted by the insertion of the recognition sequence for yeast endonuclease I-SceI. The artificially introduced 18 bp I-SceI recognition sequence was likely a unique sequence in the genome of the mouse cell line. To assess whether an induced double-strand break (DSB) in the genomic tk gene would be repaired preferentially by gene targeting or non-homologous recombination, we electroporated the mouse cell line with endonuclease I-SceI alone, one of two different gene targeting constructs alone, or with I-SceI in conjunction with each of the two targeting constructs. Each targeting construct was, in principle, capable of correcting the defective genomic tk sequence via homologous recombination. tk+ colonies were recovered following electroporation of cells with I-SceI in the presence or absence of a targeting construct. Through the detection of small deletions at the I-SceI recognition sequence in the mouse genome, we present evidence that a specific DSB can be introduced into the genome of a living mammalian cell by yeast endonuclease I-SceI. We further report that a DSB in the genome of a mouse Ltk- cell is repaired preferentially by non-homologous end-joining rather than by targeted homologous recombination with an exogenous donor sequence. The potential utility of this system is discussed.     

The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9-dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences.     

Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system.

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.     

Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells.

We have used DNA-mediated gene transfer to study homologous recombination in cultured mammalian cells. A family of plasmids with insertion and deletion mutations in the coding region of the herpes simplex type 1 thymidine kinase (tk) gene served as substrates for DNA-mediated gene transfer into mouse Ltk- cells by the calcium phosphate technique. Intermolecular recombination events were scored by the number of colonies in hypoxanthine-aminopterin-thymidine selective medium. We used supercoiled plasmids containing tk gene fragments to demonstrate that an overlap of 62 base pairs (bp) of homologous DNA was sufficient for intermolecular recombination. Addition of 598 bp of flanking homology separated from the region of recombination by a double-strand gap, deletion, or insertion of heterologous DNA increased the frequency of recombination by 300-, 20-, or 40-fold, respectively. Linearizing one of the mutant plasmids in a pair before cotransfer by cutting in the area of homology flanking a deletion of 104 bp or an insertion of less than 24 bp increased the frequency of recombination relative to that with uncut plasmids. However, cutting an insertion mutant of greater than or equal to 24 bp in the same manner did not increase the frequency. We show how our data are consistent with models that postulate at least two phases in the recombination process: homologous pairing and heteroduplex formation.     

Mechanisms of double-strand-break repair during gene targeting in mammalian cells

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.     

Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity.

Interactions between similar but not identical (homeologous) DNA sequences play an important biological role in the evolution of genes and genomes. To gain insight into the underlying molecular mechanism(s) of genetic recombination, we have studied inter- and intramolecular homeologous recombination in S. cerevisiae during transformation. We found that homeologous DNAs recombine efficiently. Hybrid sequences were obtained between two mammalian cytochrome P450 cDNAs, sharing 73% identity, and between the yeast ARG4 gene and its human homeologous cDNA, sharing 52% identity. Sequencing data showed that the preferred recombination events are those corresponding to the overall alignment of the DNA sequences and that the junctions are within stretches of identity of variable length (2-21 nt). We suggest that these events occur by a conventional homologous recombination mechanism.     

Mechanisms involved in targeted gene replacement in mammalian cells

The "ends-out" or omega (Omega)-form gene replacement vector is used routinely to perform targeted genome modification in a variety of species and has the potential to be an effective vehicle for gene therapy. However, in mammalian cells, the frequency of this reaction is low and the mechanism unknown. Understanding molecular features associated with gene replacement is important and may lead to an increase in the efficiency of the process. In this study, we investigated gene replacement in mammalian cells using a powerful assay system that permits efficient recovery of the product(s) of individual recombination events at the haploid, chromosomal mu-delta locus in a murine hybridoma cell line. The results showed that (i) heteroduplex DNA (hDNA) is formed during mammalian gene replacement; (ii) mismatches in hDNA are usually efficiently repaired before DNA replication and cell division; (iii) the gene replacement reaction occurs with fidelity; (iv) the presence of multiple markers in one homologous flanking arm in the replacement vector did not affect the efficiency of gene replacement; and (v) in comparison to a genomic fragment bearing contiguous homology to the chromosomal target, gene targeting was only slightly inhibited by internal heterology (pSV2neo sequences) in the replacement vector.