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The yeast centromere CDEI/Cpf1 complex: differences between in vitro binding and in vivo function

A Wilmen1, H Pick, R K Niedenthal

  • 1Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, Germany.

Nucleic Acids Research
|July 25, 1994
PubMed
Summary

The centromere and promoter factor Cpf1 binds yeast centromere DNA element I. Minimal binding site length is 10 base pairs, with the CACGTG core crucial for function.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Yeast Biology

Background:

  • The centromere is essential for chromosome segregation during cell division.
  • Cpf1 is a protein factor that binds to centromeric DNA elements.

Purpose of the Study:

  • To investigate the binding characteristics of Cpf1 to centromere DNA element I (CDEI).
  • To correlate in vitro Cpf1 binding with in vivo centromere function using site-directed mutagenesis.

Main Methods:

  • Site-directed mutagenesis of the CEN6-CDEI sequence in Saccharomyces cerevisiae.
  • In vitro binding assays to determine Cpf1 affinity for mutant CDEI sequences.
  • Assessment of in vivo centromere function by measuring chromosome loss rates.

Main Results:

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  • The minimal Cpf1 binding site is 10 base pairs, including CDEI and two flanking bases.
  • The palindromic core CACGTG is critical for both in vitro binding and in vivo function.
  • Symmetrical mutations in the core affect binding and function asymmetrically.
  • Enlarging the binding site increases in vitro binding but impairs in vivo function.
  • In vitro binding does not perfectly predict in vivo centromere activity.

Conclusions:

  • Cpf1 binding to CDEI is essential for centromere function in yeast.
  • The length and symmetry of the Cpf1 binding site influence its activity.
  • In vitro binding data only partially predict in vivo centromere complex behavior, suggesting additional interactions.