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Active and inactive transplacement of the M26 recombination hotspot in Schizosaccharomyces pombe
J B Virgin1, J Metzger, G R Smith
1Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
Abstract:
The ade6-M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hotspot that elevates ade6 intragenic recombination approximately 10-15-fold. A heptanucleotide sequence including the M26 point mutation is required but not sufficient for hotspot activity. We studied the effects of plasmid and chromosomal context on M26 hotspot activity. The M26 hotspot was inactive on a multicopy plasmid containing M26 embedded within 3.0 or 5.9 kb of ade6 DNA. Random S. pombe genomic fragments totaling approximately 7 Mb did not activate the M26 hotspot on a plasmid. M26 hotspot activity was maintained when 3.0-, 4.4-, and 5.9-kb ade6-M26 DNA fragments, with various amounts of non-S. pombe plasmid DNA, were integrated at the ura4 chromosomal locus, but only in certain configurations relative to the ura4 gene and the cointegrated plasmid DNA. Several integrations created new M26-independent recombination hotspots. In all cases the non-ade6 DNA was located > 1 kb from the M26 site, and in some cases > 2 kb. Because the chromosomal context effect was transmitted over large distances, and did not appear to be mediated by a single discrete DNA sequence element, we infer that the local chromatin structure has a pronounced effect on M26 hotspot activity.
Insights
The ade6-M26 mutation in fission yeast creates a recombination hotspot. Chromosomal context, not just DNA sequence, significantly influences this hotspot activity, suggesting chromatin structure plays a key role.
Area of Science:
- Genetics
- Molecular Biology
- Yeast Genetics
Background:
- The ade6-M26 mutation in Schizosaccharomyces pombe establishes a potent meiotic recombination hotspot.
- This hotspot elevates intragenic recombination approximately 10-15 fold.
- A specific heptanucleotide sequence is necessary but insufficient for hotspot function.
Purpose of the Study:
- To investigate the impact of plasmid and chromosomal contexts on the activity of the M26 recombination hotspot.
- To determine if non-ade6 DNA sequences influence M26 hotspot function.
- To elucidate the role of chromatin structure in regulating meiotic recombination hotspots.
Main Methods:
- Assessing M26 hotspot activity in different S. pombe contexts (plasmid vs. chromosome).
- Integrating M26 fragments into the ura4 locus on the chromosome.
- Analyzing recombination frequencies and identifying new hotspots in various integration configurations.
Main Results:
- The M26 hotspot was inactive on multicopy plasmids, even with extensive flanking ade6 DNA.
- Integration into the chromosome maintained M26 hotspot activity, dependent on configuration relative to ura4 and other DNA.
- Several chromosomal integrations generated novel M26-independent recombination hotspots, indicating long-range effects.
- Non-ade6 DNA located over 1-2 kb away influenced hotspot activity.
Conclusions:
- Chromosomal context, particularly local chromatin structure, profoundly impacts M26 meiotic recombination hotspot activity.
- Recombination hotspot regulation is not solely dependent on specific DNA sequences but is influenced by the genomic environment.
- The findings suggest a mechanism where chromatin influences the accessibility or function of recombination initiation sites.