Homologous Recombination Catalyzed by Human Cell Extracts

Kucherlapati, Raju; Spencer, J; Moore, Peter D.

doi:10.1128/mcb.5.4.714-720.1985

Cited by 7 publications

(3 citation statements)

References 32 publications

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“…Included among those enzymes are a 3' exonuclease, a possible DNA-pairing protein analogous to the E. coli RecA or ss DNA-binding protein, a DNA polymerase, an ss endonuclease, and a DNA ligase. With the availability of an in vitro system from mammalian cells that appears to be able to catalyze at least some of the steps in homologous recombination (9,15,17,19; N. Stemnberg and S. L. Brenner, unpublished data), it should soon be possible to test the involvement of these proteins in the recombination process. Recent experiments of Hsieh et al ( 15) and Cox and Lehman (8) have identified proteins from mammalian cells that could be RecA and ssb analogs.…”

Section: Resultsmentioning

confidence: 99%

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Extrachromosomal Recombination in Mammalian Cells as Studied with Single- and Double-Stranded DNA Substrates

Lin

Sperle

Sternberg

1987

Molecular and Cellular Biology

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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, 1984). 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 (tk delta 5' and tk delta 3'). 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.

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Section: Resultsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

Extrachromosomal Recombination in Mammalian Cells as Studied with Single- and Double-Stranded DNA Substrates

Lin

Sperle

Sternberg

1987

Molecular and Cellular Biology

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“…Homologous recombination is a mechanism implicated in fundamental processes determining genome organization. DNA double strand breaks (dsb) stimulate recombination in Escherichia coli (1), in yeast (2), in mammalian cells (3,4,5,6) as well as in cell free systems using yeast (7,8,9) or human nuclear extracts (10,11,12,13).…”

Section: Introductionmentioning

confidence: 99%

Structural effect of donor DNA on the initiation of recombination for double strand break repair in human nuclear extracts

1992

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The effect of the structure of donor DNA molecules on the initiation of recombination for double strand break repair in human nuclear extracts, was investigated here. A unique double strand break was introduced into M13 duplex derivatives by digestion with restriction enzymes. After coincubation of the cleaved DNA in human nuclear extracts, with a plasmid containing M13 sequences spanning the break, double strand break repair was estimated by the plating efficiency in JM109 (RecA1) bacteria. We first confirm that a short heterologous insert (8bp) close to the break on the recipient cleaved M13 DNA inhibits recombination with circular as well as with linear donor molecules. The results indicate that, with these substrates, recombination is initiated at the level of the break, requires uninterrupted homology on both sides of the break, and is associated with a decreasing gradient of gene conversion. When the heterologous insertion is located on the plasmid donor DNA, similar results are obtained with a circular donor DNA. In contrast, with a linear donor molecule, bearing the insert, homology requirements, in the region of the break in M13 DNA, are abolished. This last result suggests that recombination could be initiated at the extremities of the linear donor DNA.

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Characterization of Nonconservative Homologous Junctions in Mammalian Cells

Desautels

Brouillette

Wallenburg

et al. 1990

Molecular and Cellular Biology

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Homologous recombination in mammalian cells between extrachromosomal molecules, as well as between episomes and chromosomes, can be mediated by a nonconservative mechanism. It has been proposed that the key steps in this process are the generation (by double-strand cleavage) of overlapping homologous ends, the creation of complementary single-strand ends (either by strand-specific exonuclease degradation or by unwinding of the DNA helix), and finally the creation of heteroduplex DNA by the annealing of the single-strand ends. We have analyzed in detail the structure of nonconservative homologous junctions and determined the contribution of each end to the formation of the junction. We have also analyzed multiple descendants from single recombination events. Two types of junctions were found. The majority (90%) of the junctions were characterized by a single crossover site. These crossover sites were distributed randomly throughout the junction. The remaining 10% of the junctions had mosaic patterns of parental markers. Furthermore, in 9 of 10 cases, multiple descendants from a single recombination event were identical. Thus, it appears that in most cases few parental markers were involved in junction formation. This finding suggests that nonconservative homologous junctions are mediated mainly by short heteroduplexes of a few hundred base pairs or less. These results are discussed in terms of the current models of nonconservative homologous recombination.

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Homologous Recombination Catalyzed by Human Cell Extracts

Cited by 7 publications

References 32 publications

Extrachromosomal Recombination in Mammalian Cells as Studied with Single- and Double-Stranded DNA Substrates

Extrachromosomal Recombination in Mammalian Cells as Studied with Single- and Double-Stranded DNA Substrates

Structural effect of donor DNA on the initiation of recombination for double strand break repair in human nuclear extracts

Characterization of Nonconservative Homologous Junctions in Mammalian Cells

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