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Published March 15, 2010 | public
Journal Article

Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM


We have used the Xenopus laevis egg extract system to study the roles of vertebrate Dna2 in DNA replication and double-strand-break (DSB) repair. We first establish that Xenopus Dna2 is a helicase, as well as a nuclease. We further show that Dna2 is a nuclear protein that is actively recruited to DNA only after replication origin licensing. Dna2 co-localizes in foci with RPA and is found in a complex with replication fork components And-1 and Mcm10. Dna2 interacts with the DSB repair and checkpoint proteins Nbs1 and ATM. We also determine the order of arrival of ATM, MRN, Dna2, TopBP1, and RPA to duplex DNA ends and show that it is the same both in S phase and M phase extracts. Interestingly, Dna2 can bind to DNA ends independently of MRN, but efficient nucleolytic resection, as measured by RPA recruitment, requires both MRN and Dna2. The nuclease activity of Mre11 is required, since its inhibition delays both full Dna2 recruitment and resection. Dna2 depletion inhibits but does not block resection, and Chk1 and Chk2 induction occurs in the absence of Dna2.

Additional Information

© 2010 Landes Bioscience. Submitted: 12/18/09; Accepted: 12/23/09. Previously published online: www.landesbioscience.com/journals/cc/article/11049. We thank Dr. A. Eastman (Dartmouth College) for the gift of validated mirin and helpful discussions, Dr. A. Dutta (University of Virginia) for the gift of antibodies recognizing Xenopus And-1, Dr. J. Walter (Harvard University) for the gift of antibodies recognizing Xenopus Mcm10, and Dr. P. Jackson for the gift of anti-Mcm3, anti-RPA70 and anti-Cdc6 antibodies. We thank Sonja Hess of the Proteome Exploration Laboratory at Caltech for performing our mass spectrometry work, and we also thank Jules Chen for the immunofluorescence work in Figure 3. This work was supported by NIH grants GM043974 and GM070891 to W.G.D. and GM078666 to J.L.C.

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