Requirements for Ectopic Homologous Recombination in Mammalian Somatic Cells

Abstract

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 gene constant (C) region of the endogenous chromosomal immunoglobulin gene is corrected by using as a donor an ectopic wild-type C region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal 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 C 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.Homologous recombination is the process by which genetic information is exchanged between DNA duplexes and can occur by either gene conversion or crossing over. Gene conversion has the property of transferring genetic information in a nonreciprocal manner, while single reciprocal crossover leads to changes in the linkage relationship between genes or groups of genes. Homologous recombination generates new gene combinations that provide the basis for species diversification. It is thought to have played a fundamental role in genome organization and in the maintenance of homogeneity among repeated sequences and members of multigene families (8,15,16,25,56,59). As well, it can correct errors of replication and other forms of DNA damage (24, 27). However, recombination can also lead to altered gene function, altered expression of genes, or the loss of genes. These genetic alterations are implicated in the development of several human cancers (32, 46) and other abnormalities (49, 78).Our laboratory has been investigating homologous recombination in mammalian somatic (murine hybridoma) cells (3-7). Our assay detects homologous recombination between a donor wild-type immunoglobulin gene constant (C) region and the mutant C region of the haploid, chromosomal immunoglobulin gene in hybridomas which serves as the recipient sequence for recombination. Homologous recombination corrects the mutation in the recipient chromosomal gene and restores trinitrophenyl (TNP)-specific immunoglobulin M (IgM) production in mutant cells. Hybridomas making normal TNP-specific IgM are detected as plaque-forming cells (PFC) in a sensitive, TNP-specific plaque assay (4).In a previous...

A Strand Invasion 3′ Polymerization Intermediate of Mammalian Homologous Recombination

“…If a similar mechanism operates between recombining chromosomal sequences, it may preferentially lead to the generation of noncrossover recombinants (i.e., SDSA events). While formation of noncrossover (i.e., SDSA-like) recombinants has not been measured in this study, other investigators have reported a bias in noncrossover events during intra-and interchromosomal homologous recombination in yeast (Esposito 1978;Klein and Petes 1981;Haber and Hearn 1985;Christman et al 1988;Kupiec and Petes 1988;Pâques and Haber 1999;Ira et al 2003) and mammalian cells (Liskay et al 1984;Baker 1989;Shulman et al 1995;Baker et al 1996;Richardson et al 1998;Johnson and Jasin 2000). This mechanism acting at the level of recombination between chromosomal sequences would provide the means of suppressing potentially deleterious rearrangements that may arise from crossover between chromosomal sequences (Shulman et al 1995;Richardson et al 1998).…”

Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

“…Therefore, a homology length of 500 bp likely represents a MEPS (Shen and Huang 1986) that is required for the homology searching and strand invasion steps that precede the 39 extension. Interestingly, this value is lower than what has been previously established for recombination between dispersed homologous sequences, such as the 1-2 kb required for gene targeting (Shulman et al 1990;Hasty et al 1991;Thomas et al 1992) and the 2-4 kb required for ectopic homologous recombination (Baker et al 1996). Considerably less homology of between 163 and 300 bp promotes intrachromosomal recombination between closely linked sequences (Rubnitz and Subramani 1984;Liskay et al 1987;Waldman and Liskay 1988).…”

Repetitive sequence environment distinguishes housekeeping genes

“…LINE-1 elements located among high concentrations of such Alu elements might be rendered euchromatic. Since efficiency of illegitimate homologous recombination increases with length of homology (Waldman and Liskay, 1988;Baker et al, 1996), this would engender genome instability via illegitimate homologous recombination and possibly also via transposition if the element is suitably intact (Baker et al, 1996;Ostertag and Kazazian, 2001;Robertson, 2001). We would expect the genome instability engendered by euchromatic LINE-1 elements to result in these elements' being selected against.…”