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Published January 21, 2000 | public
Journal Article Open

Identification of the Xenopus laevis Homolog of Saccharomyces cerevisiae DNA2 and Its Role in DNA Replication


The DNA2 gene of Saccharomyces cerevisiae is essential for growth and appears to be required for a late stage of chromosomal DNA replication. S. cerevisiae Dna2p (ScDna2p) is a DNA helicase and also a nuclease. We have cloned and sequenced the homologous gene from Xenopus (Xenopus Dna2). Xenopus Dna2p (XDna2p) is 32% identical to ScDna2p, and the similarity extends over the entire length, including but not limited to the five conserved helicase motifs. XDna2p is even more closely related (60% identical) to a partial human cDNA. The Xenopus Dna2 (XDna2) gene was able to complement an S. cerevisiae dna2-1 mutant strain for growth at the nonpermissive temperature, suggesting that XDna2p is a functional as well as a structural homolog of the yeast protein. Recombinant XDna2p was expressed in insect cells and purified. Like the ScDna2p purified from yeast, it is a single-stranded DNA endonuclease and a DNA-dependent ATPase, suggesting that both of these activities are part of the essential function of Dna2p. However, unlike ScDna2p from yeast, recombinant XDna2p showed no DNA helicase activity. When XDna2 was immunodepleted from interphase egg extracts, chromosomal DNA replication was almost completely inhibited. From the size of the residually synthesized DNA from the XDna2-depleted egg extracts, it seems that initiation of DNA replication may be impaired. This interpretation is also supported by the normal DNA replication of M13 single-stranded DNA in the XDna2-depleted egg extracts.

Additional Information

Copyright © 2000 by the American Society for Biochemistry and Molecular Biology. (Received for publication, June 1, 1999, and in revised form, September 27, 1999) We are grateful to Dr. Phillip Carpenter and other members of Dr. William Dunphy's laboratory for guidance in establishing the Xenopus system in our laboratory. We are greatly indebted to Dr. Akiko Kumagai for cDNA from Xenopus oocytes. We thank Dr. Martin Budd for useful discussions. This work was supported by Public Health Service Grant GM25508, National Science Foundation Grant 9507352, and a Biology Division Fellowship (to Q. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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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