Mung bean nuclease was used to probe for DNA unwinding in torsionally-stressed chimeric plasmids containing two micron plasmid sequences and the yeast LEU2 gene in a pBR322 vector. The yeast sequences are cleaved at only two sites, both of which map to regulatory regions: (1) the autonomously replicating sequence (ARS), an origin of DNA replication, of the two micron plasmid and (2) the transcription terminator region of the LEU2 gene. Nucleotide level analysis of the nuclease cleavage pattern shows that an A + T-rich structure, distinct from other non-B DNA conformations, is recognized. A computer analysis reveals that A + T content alone is not sufficient to explain the preferential occurrence of the A + T-rich structure in the ARS over other sequences of equal A + T content. The A + T-rich structure detected in the ARS maps to sequences required for DNA replication. Our findings demonstrate the DNA conformational flexibility of certain yeast regulatory regions and provide support for the hypothesis that the A + T-rich sequence in the ARS plays a role in DNA unwinding during the initiation of DNA replication.

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Journal Article | Research Support, U.S. Gov't, P.H.S.

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Umek RM, Kowalski D

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