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Positioned nucleosomes inhibit Dam methylation in vivo
1Laboratory of Cellular and Developmental Biology, National Institutes of Health, Bethesda, MD 20892.
Summary
Escherichia coli Dam DNA methyltransferase (Dam) methylation of GATC sites in yeast is influenced by nucleosome positioning. Nucleosomes restrict Dam access to DNA, inhibiting methylation near the histone-DNA contact points.
Area of Science:
- Molecular Biology
- Epigenetics
- Yeast Genetics
Background:
- Escherichia coli Dam DNA methyltransferase (Dam) can methylate GATC sites in Saccharomyces cerevisiae.
- Previous studies suggested methylation levels correlate with transcriptional state or chromosomal context.
- The direct impact of nucleosome positioning on Dam methylation efficiency remained unclear.
Purpose of the Study:
- To investigate the direct influence of nucleosome location on Dam methyltransferase activity at GATC sites within yeast chromatin.
- To quantify methylation levels at specific GATC sites relative to nucleosome positioning.
Main Methods:
- Utilized yeast strains expressing Dam methyltransferase with engineered minichromosomes.
- Precisely positioned nucleosomes were analyzed within these minichromosomes.
- Quantified GATC site methylation levels using a rigorous oligonucleotide-probing procedure.
Main Results:
- GATC sites located in nucleosomal linkers and adjacent DNA (within 21 bp) were highly methylated.
- Methylation was significantly inhibited at GATC sites closer to the nucleosomal pseudodyad due to histone-DNA contacts.
- Other DNA-binding proteins were also found to interfere with Dam methylation.
Conclusions:
- Nucleosome positioning directly impacts Dam DNA methyltransferase accessibility and activity.
- Nucleosomes act as a barrier, repressing DNA modification by restricting access of trans-acting factors like Dam.
- This mechanism highlights how chromatin structure influences DNA functional regulation.