Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Abstract

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.In a number of earlier studies, we have shown that appropriately designed DNA substrates are recombined very efficiently after injection into the nuclei of large oocytes from Xenopus laevis (6,23,(33)(34)(35)39). Each oocyte has a large capacity for recombination, processing 109 substrate molecules to completed products in several hours (6). Recombination requires homology and DNA ends (6), and it appears to proceed exclusively by a nonconservative resectionannealing mechanism (also called single-strand annealing [22,35,39]). Unlike the situation in Xenopus eggs (37) and mammalian cells (42), no illegitimate end joining is observed in oocytes (6). Even products formed at low efficiency in the absence of substantial homologies depend on short sequence matches and result in small deletions (17).The resection-annealing process (Fig. 1)

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

“…Although this predominance was very pronounced and the percentage of Tef-colonies always lay between 83% and 98% (with the exception of the six hour time point showing 73%; Figure 2B), the variability in the oocytes was somewhat higher than in the eggs (Figure 2). These findings are consistent with data of D. Carroll and colleagues (Lehman et a/., 1993), where a homologous recombination activity was demonstrated to increase during oogenesis and be highly active in stage VI oocytes. Similar to the DMA-end joining activity in fertilized eggs, the final level of homologous recombination in oocytes was reached already after 20-30 min of incubation ( Figure 2B; see also Figure 4B and D) and the reaction was intramolecular (data not shown).…”

“…The significant decrease of Kan r -colonies in the 10 ng injection probably reflects an exhaustion of the highly active DNA-end joining system (see Discussion). Thus there is indeed a relatively weak homologous recombination activity observed in fertilized eggs (Lehman et a/., 1993), and it is worth pointing out that in absolute terms, we found this activity to be as high as the one in oocytes (compare numbers of Tef-colonies in Figure 3A-C to Figure 4A-C). In fertilized eggs/early embryos, homologous recombination is simply more difficult to detect because of the overwhelming efficiency of the DNA-end joining system that competes for the same substrate.…”

“…Since there was evidence for a relatively weak residual homologous recombination activity in fertilized eggs ( Figure 3D; see also Lehman et a/., 1993) we investigated the influence of the amount of injected DNA, reasoning that when the available substrate concentration was in- Both cell types exhibit similar kinetics. In oocytes the maximal level is reached after six hours (less than 3-fold), in fertilized eggs after two hours (about 2-fold).…”

Post‐Translational Activation of Non‐Homologous DNA End‐Joining in Xenopus Oocyte Extracts

“…One of the oocyte Oocyte extract was mixed with an equal volume of either unfertilised egg extract containing active maturation-promoting factor (lanes 7 -9) or interphase egg extract lacking maturation-promoting factor activity (lanes [10][11][12] in the presence of SOmg/ml cycloheximide. After incubation for 30 min at 21 "C, part of each sample was assayed for NHEJ (lanes 7,10) while the rest was mixed with an equal volume of oocyte extract (plus cycloheximide) and incubated for a further 30 min at 21 "C. Part was then assayed (lanes 8, l l ) while the remainder was diluted a third time as above.…”

A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA

“…In the last few years, the biochemistry of end-joining mechanisms has been investigated with growing interest in a variety of eucaryotic systems in vivo (Roth & Wilson 1986;Munz & Young 1991;Winegar et al 1992;Lehman et al 1993) and in vitro (Pfeiffer & Vielmetter 1988;North et al 1990;Fairman et al 1992;Derbyshire et al 1994;Nicolás & Young 1994;Lehman et al 1994). Potent end-joining activities are observed in all systems but the precise mechanisms of junction formation are different in the different in vitro systems.…”