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Published June 30, 2008 | Published + Accepted Version
Journal Article Open

Processing of G4 DNA by Dna2 Helicase/nuclease and RPA provides insights into the mechanism of Dna2/RPA substrate recognition


The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen-bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates arising in the course of DNA replication and repair. The lagging strand DNA replication protein, Dna2, has demonstrated a unique localization to telomeres and a role in de novo telomere biogenesis, prompting us to study the activities of Dna2 on G4 DNA-containing substrates. We find that yeast Dna2 binds with 25-fold higher affinity to G4 DNA formed from yeast telomere repeats than to single-stranded DNA of the same sequence. Human Dna2 also binds G4 DNAs. The helicase activities of both yeast and human Dna2 are effective in unwinding G4 DNAs. On the other hand, the nuclease activities of both yeast and human Dna2 are attenuated by the formation of G4 DNA, with the extent of inhibition depending on the topology of the G4 structure. This inhibition can be overcome by replication protein A. Replication protein A is known to stimulate the 5'- to 3'-nuclease activity of Dna2; however, we go on to show that this same protein inhibits the 3'- to 5'-exo/endonuclease activity of Dna2. These observations are discussed in terms of possible roles for Dna2 in resolving G4 secondary structures that arise during Okazaki fragment processing and telomere lengthening.

Additional Information

© 2008 the American Society for Biochemistry and Molecular Biology. Submitted on March 20, 2008. Revised on June 13, 2008. Accepted on June 30, 2008. JBC Papers in Press. Published on June 30, 2008 as Manuscript M802244200. We are grateful to Marc Wold for the gift of highly purified hsRPA and to Robert Bambara and members of his lab for discussions of the experiments. Proteins were expressed and partially purified in the Protein Expression Center at Caltech. This work was supported USPHS GM087666 and a research grant from the Philip Morris External Research Program. This work was supported, in whole or in part, by National Institutes of Health Grant GM087666 from the USPHS and a research grant from the Philip Morris External Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Accepted Version - MASjbc08pip.pdf


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