Nucleic Acids Research, 1989, Vol. 17, No. 24 10243-10258
© 1989
Articles |
Minichromosome assembly accompanying repair-type DNA synthesis in Xenopus oocytes
Department of Molecular and Experimental Medicine, Scripps Clinic and Research Foundation 10666 North Torrcy Pines Road, La Jolla, CA 92037, USA
*To whom correspondence should be addressed
Received September 26, 1989. Revised November 15, 1989. Accepted November 15, 1989.
Minichromosomes were assembled by injection of circular DNA into the nucleus of ,Xenopus oocytes. We observed that, in the course of DNA supercoiling and chromatin assembly, a small percentage of the injected DNA molecules incorporated a radioactive precursor. This DNA synthesis was carried out by aphidicolin-sensitive DNA polymerase, and generated short repair-like patches covalently linked to the injected DNA. We found that the DNA thus repaired was rapidly supercoiled almost to completion within 15 to 30 min after injection, whereas 60 to 120 min were required to supercoil the intact, bulk DNA molecules. Such differential supercoiling kinetics was also observed when UV-damaged DNA was injected. Chromatin assembly, which was characterized by DNA fragment sizes protected from micrococcal nuclease digestion, was consistent with the rapid DNA supercoiling and proceeded more efficiently on the repaired DNA. These results indicate that there are at least two kinetically distinct ways of assembling minichromosomes in the oocyte nucleus, and that the repaired DNA molecules preferentially follow the faster pathway.