Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells.

Abstract

We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination. These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site. We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination. Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference. Next, to inquire into the fate of doublestrand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer ofXho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homolog or by end-joining. Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining. These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S. cerevisiae, wherein only products of homologous events are detected.Studies using introduced, extrachromosomal DNA substrates have shown that both end-joining (nonhomologous recombination) and homologous recombination occur in mammalian somatic cells; however, the process of endjoining occurs far more frequently than homologous recombination (1). When linear simian virus 40 (SV40) genomes containing terminal homology were examined for the relative rates of nonhomologous end-joining and homologous recombination in mammalian cells, end-joining was about 3-fold more efficient than homologous recombination (2). Similarly, repair oftransfected plasmids occurs primarily by end-joining in mammalian cells (1). Restriction enzyme-induced breaks in mammalian episomal vectors are also repaired by end-joining or by religation of cohesive ends (3). Random integration generally prevails over gene-targeting (4), again suggesting a bias against homologous recombination in mammalian cells.In this report, we present a study ofthe relative efficiencies of end-joining and homologous recombination at a defined in vivo double-strand break in a mammalian chromosome. Since differences exist between extrachromosomal recombination and intrachromosomal recombination (5) as well as between gene-targeting and intrachromosomal recombination (6) in mammalian cells, we sought to determine whether the fate of chromosomal breaks differed from the fate of extrachromosomal breaks.To examine directly the relative efficiencies of end-joining and homologous recombinat...

Requirements for Ectopic Homologous Recombination in Mammalian Somatic Cells

“…The cis preference for recombination is striking, being as much as 10 4 -fold higher than recombination between the same homologous C sequences present on nonhomologous chromosomes or in their normal position on homologous chromosomes (53). A similar preference for intra-over interchromosomal recombination was also observed by us (3,7,53) and by others (19,39) when homologous repeats were closely linked in the mammalian genome. The results of the present study are significant and have important implications for our understanding of the mechanism of recombination in mammalian somatic cells.…”

Chromosome Instability as a Result of Double-Strand Breaks near Telomeres in Mouse Embryonic Stem Cells

“…The recombination junctions in the mouse ES cell subclones contained 1 to 4 bp of complementarity, while the recombination junctions in the EJ-30 subclones had 0 to 3 bp of complementarity (Table 1). In one EJ-30 subclone, a short fragment of human DNA was found inserted between the inverted plasmid sequences at the recom- (40,56,64,65,67) and insertions of short fragments of cellular DNA (23,38,40,48,56,63,67) have been previously observed during repair of DSBs by NHEJ in mammalian cells. Because the DSBs were generated at a known location with the I-SceI endonuclease in the mouse ES cell subclones, the size of the deletions occurring on the ends at the site of the break could be precisely determined (Table 1).…”

The radioprotective effect of the 24 kDa FGF-2 isoform in HeLa cells is related to an increased expression and activity of the DNA dependent protein kinase (DNA-PK) catalytic subunit

“…Accumulating evidence showed that this repair pathway is largely predominant in mammalian cells. For example, when cells are engineered with DNA chromosomal substrates that permit the repair of DSB by either homologous recombination or by NHEJ, the great majority of the joining events occurs by NHEJ (Godwin et al, 1994;Sargent et al, 1997). Secondly, the fact that HeLa 3A cells are specifically sensitized to the killing effect of IR by wortmannin also suggests a role for DNA-PK in the radioresistant phenotype.…”