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Molecular and Cellular Biology, April 1999, p. 2585-2593, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Ku-Dependent Nonhomologous DNA End Joining in Xenopus Egg Extracts

Paul Labhart*

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

Received 9 June 1998/Returned for modification 13 July 1998/Accepted 17 December 1998

An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5'-protruding single strand (PSS) and an end with a 3'-PSS, between two ends with 3'-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5'-PSS was Ku independent. These results show that the Xenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.

* Mailing address: Department of Molecular and Experimental Medicine, The Scripps Research Institute, Mail drop SBR10, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (619) 784-2406. Fax: (619) 784-2960. E-mail: plabhart{at}scripps.edu.

Molecular and Cellular Biology, April 1999, p. 2585-2593, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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