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Analysis of S. Pombe Rec12 in Meiotic Double-strand Break Formation and Removal by the Mre11-Rad50-Nbs1 Complex

出版	Université Laval, 2014
URL	http://books.google.com.hk/books?id=Ti0QuAEACAAJ&hl=&source=gbs_api

註釋Double-strand breaks are generally harmful to the cell, although there is an important exception to this rule. In yeast and other eukaryotes, Spo11 (or S. pombe Rec12) is known to create double-strand DNA breaks (DSBs), which are important to initiate meiotic recombination. Genetic and bioinformatic analyses proposed that Spo11 homologues bind double-strand DNA as a dimer and cleave the DNA by a topoisomerase-like reaction to generate transient, covalent protein-DNA intermediates. However, this view has been challenged by biochemists, but also by the fact that it is extremely difficult to purify proteins Spo11 under native conditions. To understand how Schizosaccharomyces pombe Spo11 (Rec12) generates double-strand breaks, we established a protocol for triple affinity purification. We performed in vitro assays, reminiscent of the function of Spo11 in DSB formation. The resulting purified Rec12 was pure as judged by Sypro staining. We also purified Rec12-Y98F (catalytic mutant) and Rec12-G202E (DNA binding mutant). Purified Rec12 catalysed double-strand break formation on supercoiled DNA whereas Rec12-Y98F and Rec12-G202E were inactivated. Rec12 showed activity on supercoiled DNA but not in short double-stranded DNA oligonucleotides. We mapped the possible DNA binding motifs on Rec12. Using purified mutants, we biochemically confirmed that Rec12 contains multiple DNA binding sites. Rec12 binds 5'-tailed DNA but not 3'-tailed DNA, which is reminiscent of the accumulation of Spo11 on meiotic DNA double-strand breaks. We also analyzed biochemically how Rec12 is removed from DSBs by the Rad32 (Mre11) nuclease to produce single-strand overhang that can undergo DNA recombination. Some of the results presented were also confirmed using human and S. cerevisiae Spo11. We also discuss how endo- and exonuclease activities of MRE11-RAD50-NBS1 (MRN) complex regulates double-strand break repair pathway choice. By using structure-based designed MRE11 endo- or exonuclease inhibitors we represented here specific nuclease roles in DSB repair. Altogether, our results indicate that Rec12 is responsible for the formation of double-strand breaks, an activity that is regulated by multiple DNA binding sites. This study represents a first glimpse of the biochemical function of yeast or human Spo11 under native conditions.