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Nucleic Acids Research, 1990, Vol. 18, No. 22 6601-6605
© 1990

MOLECULAR BIOLOGY

The DNA unwinding element in a yeast replication origin functions independently of easily unwound sequences present elsewhere on a plasmid

Robert M. Umek+ and David Kowalski*

Department of Molecular and Cellular Biology, Roswell Park Cancer Institute Buffalo, NY 14263, USA

* To whom correspondence should be addressed

Received July 19, 1990. Revised October 10, 1990. Accepted October 10, 1990.

We have previously identified a DNA unwinding element (DUE) in autonomously replicating sequences (ARSs) and demonstrated a correlation between single-strand-specific nuclease hypersensitivity of the DUE and ARS-mediated plasmid replication in yeast. The DUE in the H4 ARS is the most easily unwound sequence in a supercolled DNA molecule, in the context of the Ylp5 plasmid. To determine whether sequences which are more readily unwound than the ARS can influence replication activity, we have inserted such sequences, called ‘torsional sinks’, into the plasmids at a site distal to the ARS. We show that the torsional sink sequences effect reduction or elimination of the nuclease hypersensitivity of a variety of H4 ARS derivatives. However, we detect no difference in the in vivo replication activity of an individual ARS plasmid with or without a torsional sink. Thus, the function of the DUE in a yeast replication origin is unaffected by easily unwound sequences present elsewhere on the same plasmid.

+ Present address: Carnegie Institution of Washington, Department of Embryology, Baltimore, MD 21210, USA


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