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Characterization of the Endonuclease and ATP-dependent Flap Endo/Exonuclease of Dna2

Fortini, Barbara K. and Pokharel, Subhash and Polaczek, Piotr and Balakrishnan, Lata and Bambara, Robert A. and Campbell, Judith L. (2011) Characterization of the Endonuclease and ATP-dependent Flap Endo/Exonuclease of Dna2. Journal of Biological Chemistry, 286 (27). pp. 23763-23770. ISSN 0021-9258. PMCID PMC3129157.

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Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5′ single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5′ single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn^(2+). The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity.

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Additional Information:© 2011 American Society for Biochemistry and Molecular Biology, Inc. Received for publication, March 23, 2011, and in revised form, May 4, 2011; Published, JBC Papers in Press, May 13, 2011. This work was supported, in whole or in part, by National Institutes of Health Grants GM024441 (to R. A. B.) and GM078666 (to J. L. C.). This work was also supported by Army Research Office Grant ARO09-1-0041 and Ellison Foundation Grant AG-55-2143 (to J. L. C.).
Funding AgencyGrant Number
Army Research Office (ARO)ARO-09-1-0041
Ellison FoundationAG-55-2143
Issue or Number:27
PubMed Central ID:PMC3129157
Record Number:CaltechAUTHORS:20110718-082346640
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Official Citation:Characterization of the Endonuclease and ATP-dependent Flap Endo/Exonuclease of Dna2 Barbara K. Fortini, Subhash Pokharel, Piotr Polaczek, Lata Balakrishnan, Robert A. Bambara, and Judith L. Campbell J. Biol. Chem. 2011 286: 23763-23770. First Published on May 13, 2011, doi:10.1074/jbc.M111.243071
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:24439
Deposited By: Ruth Sustaita
Deposited On:18 Jul 2011 16:11
Last Modified:03 Oct 2019 02:56

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