Homologous recombination during meiosis mediates reductional segregation of chromosomes and increases genetic diversity among progeny. Errors in meiotic recombination can result in miscarriages, congenital birth defects, and infertility. Hotspots regulate the frequency and position of recombination events during meiosis, but their mechanism of activation remains poorly understood. Current models of hotspot activation involve post-translational modifications of histones and recruitment of chromatin remodeling enzymes to the hotspot. To test these models and to identify novel regulators of meiotic recombination hotspots, an affinity-capture approach was developed and implemented to purify the ade6-M26 meiotic recombination hotspot from fission yeast and quantify proteins associated with this locus using mass spectrometry.
Small, circular minichromosomes containing the ade6-M26 hotspot or control locus were constructed and transformed into fission yeast strains and purified from synchronous meiotic cultures. Analysis of the large-scale data sets and validation experiments revealed five novel regulators of the ade6-M26 meiotic recombination hotspot. The histone chaperones Hir1 and Nap1, the INO80 complex subunits Arp5 and Arp8, and the ATP-dependent chromatin remodeling enzyme Fft3 contribute to hotspot activation through distinct, possibly overlapping mechanisms. More than 30 unique, combinatorial histone post-translational modifications were detected and quantified between hotspot and control throughout meiosis. Overall, this dissertation work demonstrates the utility of affinity purification experiments coupled to mass spectrometry to reveal novel insights into the regulation of crucial biological processes, and suggests a conserved mechanism of hotspot activation through the interplay between these protein complexes and the H2A.Z histone variant.
|Advisor:||Wahls, Wayne P.|
|Commitee:||Davidson, Mari K., Eoff, Robert L., Haun, Randy S., Tackett, Alan J.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 79/10(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||Hotspots, Mass spectrometry, Meiosis, Recombination|
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