Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes

1986 ◽  
Vol 6 (6) ◽  
pp. 2053-2061
Author(s):  
D Carroll ◽  
S H Wright ◽  
R K Wolff ◽  
E Grzesiuk ◽  
E B Maryon
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
Xenopus Oocyte ◽  
Double Strand Breaks ◽  
Xenopus Laevis Oocytes ◽  
Strand Breaks ◽  
Linear Dna ◽  
Linear Molecules ◽  
Dna Ends ◽  
Detailed Molecular Analysis

When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other. With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval. With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra- and intermolecular events take place very efficiently with linear molecules. As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed. Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions. These observations are combined to evaluate possible models of recombination in the oocytes. Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.

scholarly journals Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

1986 ◽  
Vol 6 (6) ◽  
pp. 2053-2061 ◽  
Author(s):  
D Carroll ◽  
S H Wright ◽  
R K Wolff ◽  
E Grzesiuk ◽  
E B Maryon
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
Xenopus Oocyte ◽  
Double Strand Breaks ◽  
Xenopus Laevis Oocytes ◽  
Strand Breaks ◽  
Linear Dna ◽  
Linear Molecules ◽  
Dna Ends ◽  
Detailed Molecular Analysis

When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other. With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval. With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra- and intermolecular events take place very efficiently with linear molecules. As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed. Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions. These observations are combined to evaluate possible models of recombination in the oocytes. Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes

1990 ◽  
Vol 10 (11) ◽  
pp. 5849-5856
Author(s):  
S E Sweigert ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Coli Strain ◽  
Xenopus Laevis Oocytes ◽  
X Rays ◽  
Strand Breaks ◽  
X Ray ◽  
Dna Strand

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.

scholarly journals Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes.

1989 ◽  
Vol 9 (11) ◽  
pp. 4862-4871 ◽  
Author(s):  
E Maryon ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
High Efficiency ◽  
Average Rate ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Exonuclease Activity ◽  
Linear Dna ◽  
Homologous Sequences

Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'----3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei.

Homologous and illegitimate recombination in developing Xenopus oocytes and eggs

1993 ◽  
Vol 13 (11) ◽  
pp. 6897-6906
Author(s):  
C W Lehman ◽  
M Clemens ◽  
D K Worthylake ◽  
J K Trautman ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Developmental Stages ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Xenopus Laevis Oocytes ◽  
Recombination Activity ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Nuclear Extracts ◽  
Reaction Proceeds

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.

scholarly journals Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes.

1990 ◽  
Vol 10 (11) ◽  
pp. 5849-5856 ◽  
Author(s):  
S E Sweigert ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Coli Strain ◽  
Xenopus Laevis Oocytes ◽  
X Rays ◽  
Strand Breaks ◽  
X Ray ◽  
Dna Strand

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.

Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes

1989 ◽  
Vol 9 (11) ◽  
pp. 4862-4871
Author(s):  
E Maryon ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
High Efficiency ◽  
Average Rate ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Exonuclease Activity ◽  
Linear Dna ◽  
Homologous Sequences

Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'----3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei.

scholarly journals Switch-Like Phosphorylation of Wrn Integrates End-Resection With Repair of Dsbs at Replication Forks

2018 ◽  
Author(s):  
Valentina Palermo ◽  
Eva Malacaria ◽  
Massimo Sanchez ◽  
Annapaola Franchitto ◽  
Pietro Pichierri
Keyword(s):  
Homologous Recombination ◽  
Long Range ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Strand Invasion ◽  
Wrn Helicase ◽  
End Resection ◽  
Atr Kinases ◽  
Dna Ends

ABSTRACTReplication-dependent DNA double-strand breaks are harmful lesions preferentially repaired by homologous recombination, a process that requires processing of DNA ends to allow RAD51-mediated strand invasion. End-resection and subsequent repair are two intertwined processes, but the mechanism underlying their execution is still poorly appreciated. The WRN helicase is one of the crucial factors for the end-resection and is instrumental to select the proper repair pathway. Here, we reveal that ordered phosphorylation of WRN by the CDK1, ATM and ATR kinases define a complex regulatory layer that is essential for correct long-range end-resection connecting it to repair by homologous recombination. We establish that long-range end-resection requires an ATM-dependent phosphorylation of WRN at Ser1058 and that phosphorylation at Ser1141, together with dephosphorylation at the CDK1 site Ser1133, is needed to conclude long-range end-resection and support RAD51-dependent repair. Collectively, our findings suggest that regulation of WRN by multiple kinases functions as molecular switch to allow a timely execution of end-resection and repair at replication-dependent DNA double-strand breaks.

scholarly journals Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

1993 ◽  
Vol 13 (11) ◽  
pp. 6897-6906 ◽  
Author(s):  
C W Lehman ◽  
M Clemens ◽  
D K Worthylake ◽  
J K Trautman ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Developmental Stages ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Xenopus Laevis Oocytes ◽  
Recombination Activity ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Nuclear Extracts ◽  
Reaction Proceeds

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.

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