A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs

Background: Alteration of chromatin structure is a key step in various aspe cts of DNA metabolism. DNA unwinding factors such as the high mobility grou p (HMG) proteins are thought to play a general role in controlling chromati n structure and a specific role in controlling DNA replication. For instanc e, in the in vitro simian virus 40 replication system, minichromosomes cont aining HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unf olding the higher-order chromatin structure. At present, however, only limi ted data suggest an involvement of DNA unwinding factors in DNA replication , Results: We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptid es, DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce ne gative supercoils into DNA at the expense of ATP hydrolysis. Cloning and se quencing of the cDNAs encoding the two polypeptides revealed that the 87 kD a polypeptide is homologous to a mammalian HMG protein, T160/structure-spec ific recognition protein. The 140 kDa polypeptide is homologous to yeast Cd c68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the ext ract to replicate exogenously added sperm chromatin or plasmid DNA, Conclusions: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.