Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

Brouillette, S; Chartrand, Pascal

doi:10.1128/mcb.7.6.2248

Cited by 30 publications

(21 citation statements)

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“…Supporting the latter view, crosstalk between HR and NHEJ has been described, suggesting overlaps during the cell cycle. The two processes can compete or in contrast be coupled for DSB repair (Brouillette and Chartrand, 1987;Belmaaza et al, 1990;Belmaaza and Chartrand, 1994;Richardson and Jasin, 2000a;Pierce et al, 2001;Allen et al, 2002;Delacote et al, 2002). Consequently, the over-stimulation of HR in NHEJdefective cells (Pierce et al, 2001;Allen et al, 2002;Delacote et al, 2002) could easily be explained by defects in NHEJ in G2.…”

Section: Introductionmentioning

confidence: 99%

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

et al. 2006

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Non-homologous end joining (NHEJ) and homologous recombination (HR) are two pathways that can compete or cooperate for DNA double-strand break (DSB) repair. NHEJ was previously shown to act throughout the cell cycle whereas HR is restricted to late S/G2. Paradoxically, we show here that defect in XRCC4 (NHEJ) leads to over-stimulation of HR when cells were irradiated in G1, not in G2. However, XRCC4 defect did not modify the strict cell cycle regulation for HR (i.e. in S/G2) as attested by (i) the formation of Rad51 foci in late S/G2 whatever the XRCC4 status, and (ii) the fact that neither Rad51 foci nor HR (gene conversion plus single-strand annealing) events induced by ionizing radiation were detected when cells were maintained blocked in G1. Finally, both c-H2AX analysis and pulse field gel electrophoresis showed that following irradiation in G1, some DSBs reached S/G2 in NHEJ-defective cells. Taken together, our results show that when cells are defective in G1/S arrest, DSB produced in G1 and left unrepaired by XRCC4 can be processed by HR but in late S/G2.

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Section: Introductionmentioning

confidence: 99%

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

et al. 2006

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“…One is conservative and is most readily explained by the double-strand break repair model (28), while the other is nonconservative and is best explained by the single-strand annealing model (16). The most direct evidence of nonconservative homologous recombination is the observation that intermolecular recombination events can produce molecules with one homologous junction plus one nonhomologous junction that has lost the reciprocal homologous sequences (4,25). Nonconservative homologous recombination has been readily observed in extrachromosomal (2,4,6,18,25) as well as episomechromosome (24) recombination assays.…”

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confidence: 99%

“…The most direct evidence of nonconservative homologous recombination is the observation that intermolecular recombination events can produce molecules with one homologous junction plus one nonhomologous junction that has lost the reciprocal homologous sequences (4,25). Nonconservative homologous recombination has been readily observed in extrachromosomal (2,4,6,18,25) as well as episomechromosome (24) recombination assays. The molecular intermediates and enzymes involved in this recombination pathway have not yet been identified.…”

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Characterization of nonconservative homologous junctions in mammalian cells.

et al. 1990

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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 singlestrand 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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“…Before chromosomal integration takes place, the injected DNA molecules recombine to large tandemly arranged concatemers. Although the dominant intermolecular recombination mechanism in mammalian cells is non-homologous, the predominant head-to-tail array strongly suggests a process of homologous recombination between multiple copies (Brouillette & Chartrand, 1987;Bishop & Smith, 1989). The integration of pronuclear injected DNA was demonstrated by in situ hybridization in diverse regions of the mouse genome (Michalova et al 1988;Festenstein et al 1996;Dobie et al 1996;Boyer et al 1997).…”

Section: Integration Characteristics Of the Pronuclear Injected Transmentioning

confidence: 99%

Germ line transformation of mammals by pronuclear microinjection

Rülicke¹,

Hübscher²

2000

Exp. Physiol.

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Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

Cited by 30 publications

References 54 publications

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

Characterization of nonconservative homologous junctions in mammalian cells.

Germ line transformation of mammals by pronuclear microinjection

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