Involvement of Single-Stranded Tails in Homologous Recombination of DNA Injected into Xenopus laevis Oocyte Nuclei

Abstract

Homologous recombination of DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient when those molecules are linear and have overlapping homologous ends. It was previously shown that a 5′→3′ exonuclease activity in oocytes attacks injected linear DNAs and leaves them with single-stranded 3′ tails. We tested the hypothesis that such tailed molecules are early intermediates on the pathway to recombination products. Substrates with 3′ tails were made in vitro and injected into oocytes, where they recombined rapidly and efficiently. In experiments with mixed substrates, molecules with 3′ tails entered recombination intermediates and products more rapidly than did molecules with flush ends. Molecules endowed in vitro with 5′ tails also recombined efficiently in oocytes, but their rate was not faster than for flush-ended substrates. In most cases, the 5′ tails served as templates for resynthesis of the 3′ strands, regenerating duplex ends which then entered the normal recombination pathway. In oocytes from one animal, some of the 5′ tails were removed, and this was exacerbated when resynthesis was partially blocked. Analysis by two-dimensional gel electrophoresis of recombination intermediates from 5′-tailed substrates confirmed that they had acquired 3′ tails as a result of the action of the 5′→3′ exonuclease. These results demonstrate that homologous recombination in oocytes proceeds via a pathway that involves single-stranded 3′ tails. Molecular models incorporating this feature are discussed.