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 appea rs to be required for a late stage of chromosomal DNA replication. S. cerev isiae Dna2p (ScDna2p) is a DNA helicase and also a nuclease. We have cloned and sequenced the homologous gene from Xenopus (Xenopus Dnaa), Xenopus Dna 2p (XDna2p) is 32% identical to ScDna2p, and the similarity extends over th e entire length, including but not limited to the five conserved helicase m otifs, XDna2p is even more closely related (60% identical) to a partial hum an cDNA. The Xenopus Dnaa (XDna2) gene was able to complement an S, cerevis iae dna2-1 mutant strain for growth at the nonpermissive temperature, sugge sting that XDna2p is a functional as well as a structural homolog of the ye ast protein. Recombinant XDna2p was expressed in insect cells and purified. Like the ScDna2p purified from yeast, it is a single-stranded DNA endonucl ease and a DNA-dependent ATPase, suggesting that both of these activities a re part of the essential function of Dna2p. However, unlike ScDna2p from ye ast, recombinant XDna2p showed no DNA helicase activity. When XDna2 was imm unodepleted from interphase egg extracts, chromosomal DNA replication was a lmost completely inhibited. From the size of the residually synthesized DNA from the XDna2-depleted egg extracts, it seems that initiation of DNA repl ication may be impaired, This interpretation is also supported by the norma l DNA replication of M13 single-stranded DNA in the XDna2-depleted egg extr acts.