Purification, folding, and characterization of Rec12 (Spo11) meiotic recombinase of fission yeast.
Meiotic recombination is initiated by controlled dsDNA breaks (DSBs). Rec12 (Spo11) protein of fission yeast is essential for the formation of meiotic DSBs in vivo, for meiotic recombination, and for segregation of chromosomes during meiosis I. Rec12 is orthologous to Top6A topoisomerase of Archaea and is likely the catalytic subunit of a meiotic recombinase that introduces recombinogenic DSBs. However, despite intensive effort, it has not been possible to produce Rec12 protein in a soluble form required to permit biochemical analyses of function. To obtain purified Rec12 protein for in vitro studies, a rec12(+) cDNA was generated, cloned into vector pET15b(+), and expressed in Escherichia coli. Rec12 protein was produced at moderate levels and it partitioned into insoluble fractions of whole-cell extracts. The protein was enriched based upon its differential solubility in two different denaturants and was further purified by column chromatography. A combinatorial, fractional, factorial approach was used to identify conditions under which Rec12 protein could be refolded. Four parameters were most important and, following optimization, soluble Rec12 protein was obtained. Gel filtration demonstrated that refolded Rec12 protein exists as a monomer in solution, suggesting that additional proteins may be required to assemble biologically-active Rec12 dimers, as inferred previously from genetic data [Cell Chromosome 1 (2002) 1]. The production of refolded Rec12 in a soluble form will allow for characterization in vitro of this key meiotic recombination enzyme.
Wu
Heng
H
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Gao
Jun
J
Sharif
Wallace D
WD
Davidson
Mari K
MK
Wahls
Wayne P
WP
D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008540:Meiosis; D008957:Models, Genetic; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000099257:Meiotic Recombination Protein SPO11
D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C456270:rec12 protein, S pombe; D000099257:Meiotic Recombination Protein SPO11