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Related Experiment Videos

Initiation preference at a yeast origin of replication

B J Brewer1, W L Fangman

  • 1Department of Genetics, University of Washington, Seattle 98195.

Proceedings of the National Academy of Sciences of the United States of America
|April 12, 1994
PubMed
Summary
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Replication origins in yeast, called autonomous replication sequence (ARS) elements, show interference when closely spaced. Plasmid context and specific DNA sequences influence which ARS is preferentially used for replication initiation.

Area of Science:

  • Molecular Biology
  • Yeast Genetics
  • DNA Replication

Background:

  • Replication origins in Saccharomyces cerevisiae are known as autonomous replication sequence (ARS) elements.
  • Understanding origin density is crucial for studying DNA replication initiation and control.

Purpose of the Study:

  • To investigate the impact of origin density on replication initiation.
  • To analyze the replication of a plasmid containing two identical ARS1 origins.

Main Methods:

  • Utilized two-dimensional agarose gel electrophoresis to analyze replication intermediates.
  • Examined replication fork direction and origin activation.
  • Performed deletion analysis to identify sequences responsible for origin preference.

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Main Results:

  • Identical ARS elements show interference when closely spaced, with only one initiating replication per molecule.
  • One ARS1 copy was preferentially used four times more often than the other in the constructed plasmid.
  • This origin preference was maintained when ARS1 copies were integrated into a yeast chromosome.

Conclusions:

  • Close proximity of ARS elements leads to apparent interference, influencing replication initiation.
  • Plasmid and chromosomal context critically determine preferred origin usage.
  • Specific sequences within the URA3 gene are involved in establishing origin preference.