Related Experiment Videos
Unwound regions in yeast centromere IV DNA
1Department of Cell Biology, Weizmann Institute of Science, Rehovet, Israel.
Journal of Molecular Biology
|October 21, 1994
Summary
The centromere DNA of yeast chromosome IV (CEN4) unwinds in supercoiled plasmids, forming a paranemic intermediate. This structure, sensitive to nuclease P1, reveals unique DNA conformations critical for centromere function.
Area of Science:
- Molecular Biology
- Yeast Genetics
- DNA Structure
Background:
- Centromeres are crucial for chromosome segregation during cell division.
- The centromere DNA (CEN4) of Saccharomyces cerevisiae contains essential elements (CDEI, CDEII, CDEIII).
- DNA structure and conformation can influence protein binding and function.
Purpose of the Study:
- To investigate the structural properties of CEN4 DNA in a supercoiled plasmid.
- To identify regions of CEN4 DNA sensitive to single-strand specific nuclease P1.
- To understand the DNA conformation adopted by CEN4 under superhelical stress.
Main Methods:
- Treatment of supercoiled plasmids containing CEN4 with single-strand specific nuclease P1.
- Fine mapping of nuclease cleavage sites.
- Modification with DNA-confirmation-specific reagent KMnO4.
- Two-dimensional topoisomer analysis.
Main Results:
- Two P1-sensitive segments were identified within CEN4, one in CDEI/CDEII and another in CDEIII.
- The AT-rich CDEII region was largely resistant to P1 nuclease.
- Negative superhelicity was required for P1 and KMnO4 modifications.
- Two-dimensional topoisomer analysis indicated unwinding of approximately 80 bases in the CEN4 region.
Conclusions:
- Supercoiled CEN4 DNA transitions into a paranemic intermediate structure.
- This intermediate involves a foldback structure of each DNA strand.
- The identified sensitive regions and structural transition are relevant to centromere function.