Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates.

Abstract

We have previously proposed a model to account for the high levels of homologous recombination that can occur during the introduction of DNA into mammalian cells (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034). An essential feature of that model is that linear molecules with ends appropriately located between homologous DNA segments are efficient substrates for an exonuclease that acts in a 5'-)3' direction. That process generates complementary single strands that pair in homologous regions to produce an intermediate that is processed efficiently to a recombinant molecule. An alternative model, in which strand degradation occurs in the 3'-+5' direction, is also possible. In this report, we describe experiments that tested several of the essential features of the model. We first confirmed and extended our previous results with double-stranded DNA substrates containing truncated herpesvirus thymidine kinase (tk) genes (tkA5' and tkA3'). The results illustrate the importance of the location of double-strand breaks in the successful reconstruction of the tk gene by recombination. We next transformed cells with pairs of single-stranded DNAs containing truncated tk genes which should anneal in cells to generate the recombination intermediates predicted by the two alternative models. One of the intermediates would be the favored substrate in our original 5'-3' degradative model and the other would be the favored substrate in the alternative 3'--5' degradative model. Our results indicate that the intermediate favored by the 3'--5' model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.DNA introduced into cultured mammalian cells, either as a calcium phosphate precipitate or by direct microinjection into nuclei, is capable of undergoing efficient homologous recombination before it becomes integrated into chromosomes (11,13,18,22,28,32,34,35,41,42). Frequencies of homologous recombination in the range of 10 to 20% are common in these sorts of experiments (22). Recent in vitro recombination studies with nuclear extracts indicate that the enzymes necessary to execute homologous recombination are indeed present in mammalian cells (9,15,17,19).Two general mechanistic models have been proposed to account for the process of recombination during the introduction of DNA into mammalian cells. One, the doublestrand (ds)-break repair model, is analogous to that proposed to explain homologous recombination in yeasts (37). The second, the single-strand (ss) annealing model (22), is analogous to a model originally proposed by Broker and Lehman (4) to explain recombination by bacteriophage T4 and one later proposed by Lai and Nathans (20) to explain the generation of deletions after the introduction of simian virus 40 DNA into mammalian cells. In the ds-break repair model, recombination is initiated by a ds break or gap in one DNA molecule which is repaired faithfully with a second unbroken copy of the homologous DNA as a template. A requirement of the model is th...

Triplex-induced Recombination in Human Cell-free Extracts

“…Note that this effect occurred in the extract and was not mediated by recombination in the indicator bacteria because, without incubation in the extract, no recombinant products were observed upon transformation of the AϪAG30 sample into bacteria. The A donor fragment was also somewhat active, as co-mixture of A plus the pSupFG1/G144C plasmid led to a low level of supFG1 reversion, consistent with the ability of short fragments of DNA to mediate recombination and marker rescue (31)(32)(33). However, the effect of AϪAG30 was 4-fold higher than that of A alone, demonstrating the influence of the TFO domain and providing direct evidence for triplex-induced recombination in vitro.…”

Transcription of a Donor Enhances Its Use during Double-Strand Break-Induced Gene Conversion in Human Cells

“…A bias toward proximal donors is more likely when donors are separated by larger distances; such a bias would serve to stabilize the genome by reducing the probability of interactions over large distances and the associated risk of large-scale deletions and rearrangements. In both yeast and mammalian cells, SSA efficiency depends on the timing with which complementary single strands are exposed in direct repeats (17,35,36,59). Given that crossovers are restricted, most distal-proximal deletions probably arise by SSA, and the lack of bias in favor of proximal deletions suggests that end processing is rapid and extensive, exposing the relatively 32 P-labeled neo probe; the autoradiograph is shown above; equal loading is shown by ethidium bromide staining of 28S rRNA (B) Percentage of gene conversion events using 5Ј donor as a repair template for 5Ј3Ј, the net value for 5Ј3Ј and 5Ј3ЈSwitch, and MMTV5Ј3Ј Ϯ dex.…”

Illegitimate DNA integration in mammalian cells

“…Homologous recombination can modify extrachromosomal DNA very efficiently using either inter-or intramolecular sequence homology and can result in multiple products depending on the organization of the sequences sharing identity. [21][22][23][24][25][26] The end products of extrachromosomal homologous recombination are similar to those obtained during chromosomal homologous recombination, suggesting that at least some mechanisms are shared by the two processes. [27][28][29][30][31] Transfected DNA can also be mutated at high frequency (on the order of 1%) by homology-independent means including point mutations, deletions, and more complex rearrangements such as insertion of genomic DNA.…”