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Proteomic analysis of a meiotic recombination hotspot
Author(s) -
Storey Aaron J,
Protacio Reine U,
Tackett Alan J,
Davidson Mari K,
Wahls Wayne P
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.792.2
Subject(s) - homologous recombination , histone , biology , genetics , meiosis , chromatin , dna , microbiology and biotechnology , gene
Meiotic recombination promotes genetic diversity within species and is essential for accurate segregation of homologous chromosomes in meiosis I. Hotspots regulate the frequency and distribution of recombination in the genome. DNA binding proteins and post‐translational modifications (PTMs) of histones help to regulate hotspots by recruiting the basal recombination machinery (e.g., Spo11/Rec12), but few of the ~400 known histone PTMs have even been interrogated for a potential role in recombination. Here, we developed a mass spectrometry‐based approach for the unbiased, systematic discovery of hotspot‐specific (potentially regulatory) proteins and histone PTMs using the well‐characterized, DNA sequence‐dependent hotspot ade6‐M26 of fission yeast. We constructed small (4.2 kbp) minichromosomes (MiniCs) harboring hotspot and negative control alleles. Chromatin immunoprecipitation (ChIP) of recombination‐initiating dsDNA breaks revealed that the hotspot is active in MiniCs. MiniCs were purified from meiotic cultures using the high‐affinity interaction between LacO DNA sites in the MiniCs and a prA‐LacI fusion protein conjugated to beads. Enrichment levels of 20,000‐fold (relative to single copy loci) were routinely observed by gel electrophoresis and quantitative PCR. Mass spectrometry of the MiniCs identified 1023 proteins, 71 of which were enriched specifically in the hotspot sample. Fifteen different histone PTMs were identified, along with 7 histone modifying enzymes and 37 recombination‐related proteins. The DNA repair proteins Dmc1 and Rad50 were highly enriched in the hotspot sample, further validating the sensitivity and accuracy of this approach. We are currently implementing isobaric mass tag strategies using the latest generation of mass spectrometry instruments for more powerful quantitation of resulting proteomic datasets, and we are validating the biological functions of newly discovered hotspot factors. Support or Funding Information National Institute of General Medical Sciences (R01 GM081766)