Current Biology
Volume 9, Issue 7, 8 April 1999, Pages 341-351
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Research Paper
A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs

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Abstract

Background: Alteration of chromatin structure is a key step in various aspects of DNA metabolism. DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication. For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure. At present, however, only limited 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 polypeptides. 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 negative supercoils into DNA at the expense of ATP hydrolysis. Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein. The 140 kDa polypeptide is homologous to yeast Cdc68, 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 extract to replicate exogenously added sperm chromatin or plasmid DNA.

Conclusions: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.

Cited by (0)

K Okuhara (corresponding author), Laboratory of Cellular and Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0106 and Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, 113-0033, Japan. Present address: Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. e-mail: [email protected].

K Ohta, Y Tanaka and T Shibata, Laboratory of Cellular and Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0106, Japan.

H Seo and T Yamada, Laboratory of Cellular and Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0106 and Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.

M Shioda, Department of Biology, Faculty of Science, Kumamoto University, Kumamoto 860, Japan.

N Dohmae, Division of Biomolecular Characterization, Department of Research Fundamentals Technology, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0106, Japan.

Y Seyama, Department of Physiological Chemistry and Metabolism, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.

H Murofushi, Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.