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DNA double-strand breaks induce formation of RP-A/Ku foci on in vitro reconstituted Xenopus sperm nuclei

Paola Grandi^1,*,, Michail Eltsov^2,3,*,, Inga Nielsen¹ and Ivan Raka^2,3,¶

¹ Department of Biochemistry and Molecular Biology, University of Geneva, CH1211-Geneva 4, Switzerland
² Department of Cell Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Albertov 4, CZ-128 00 Prague 2, Czech Republic
³ Laboratory of Gene Expression, 1st Faculty of Medicine, Charles University, Albertov 4, CZ-128 00 Prague 2, Czech Republic
^* These authors contributed equally to this study
Present address: CellZome, Meyerhofstrasse 1, 69117, Heidelberg, Germany
On leave from: Institute of Cytology, Academy of Sciences of the Russian Federation, St Petersburg, Russian Federation

¶Author for correspondence (e-mail: iraska{at}lf1.cuni.cz)

Accepted June 10, 2001

Replication protein A (RP-A) is involved in DNA replication, repair and recombination. It has been demonstrated that RP-A clusters in foci prior to DNA replication and redistributes over chromatin during S-phase. Here, we show that RP-A foci also form in response to DNA double-strand (ds) breaks produced on Xenopus laevis sperm nuclei by restriction enzymes and then reconstituted with Xenopus egg high-speed extracts. Ku86 co-localizes with RP-A in the same foci. An unscheduled RP-A-dependent DNA synthesis takes place overlapping with RP-A and Ku86 foci. Immunoelectron-microscopy analysis reveals that these foci correspond to spherical bodies up to 300 nm in diameter, which contain RP-A, Ku86 and DNA. In an independent in vitro assay, we incubated linear dsDNA bound to magnetic beads with Xenopus egg extracts. Here, also RP-A and Ku cluster in foci as seen through immunofluorescence. Both proteins appear to enrich themselves in sequences near the ends of the DNA molecules and influence ligation efficiency of ds linear DNA to these ends. Thus, the Xenopus in vitro system allows for the generation of specific DNA ds breaks, RP-A and Ku can be used as markers for these lesions and the repair of this type of DNA damage can be studied under conditions of a normal nuclear environment.

Key words: Reconstituted Xenopus sperm nuclei, DNA double-strand breaks, Replication protein A/Ku protein foci, Unscheduled DNA synthesis

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