Homologous recombination catalyzed by a nuclear extract from Xenopus oocytes.
Xenopus laevis oocytes efficiently recombine linear DNA injected into their nuclei (germinal vesicles). This process requires homologous sequences at or near the molecular ends. Here we report that a cell-free extract made from germinal vesicles is capable of accomplishing the complete recombination reaction in vitro. Like the in vivo process, the extract converts the overlapping ends of linear substrate molecules into covalently closed products. Establishment of this cell-free system has allowed examination of the cofactors required for recombination. The first step involves a 5'----3' exonuclease activity that requires a divalent cation but not NTPs. Completion of recombination requires a hydrolyzable NTP; maximal product formation occurs in the presence of millimolar levels of ATP or dATP. At submillimolar levels of all four dNTPs, homologous recombination is inefficient, and a side reaction produces end-joined products. This cell-free system should facilitate a step-by-step understanding of an homologous recombination pathway that operates not only in Xenopus laevis oocytes but also in cells from a wide variety of organisms.
Lehman
C W
CW
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Carroll
D
D
D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D002467:Cell Nucleus / Q000502:physiology*; D004269:DNA, Bacterial / Q000235:genetics; D003854:Deoxyribonucleotides / Q000378:metabolism; D005260:Female; D009865:Oocytes / Q000502:physiology*; D010957:Plasmids; D011995:Recombination, Genetic / Q000187:drug effects; D015183:Restriction Mapping; D014020:Tissue Extracts / Q000494:pharmacology; D014982:Xenopus laevis
D000227:Adenine Nucleotides; D004269:DNA, Bacterial; D003854:Deoxyribonucleotides; D014020:Tissue Extracts