Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR 72205-7199, USA.
Nucleic Acids Res. 2011 Mar;39(4):1460-72. doi: 10.1093/nar/gkq993. Epub 2010 Oct 28.
In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein-protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics.
在裂殖酵母和其他真核生物中，Rec12（Spo11）被认为催化双链 DNA 断裂（DSB）的形成，从而启动减数分裂中的同源重组。Rec12 与拓扑异构酶 VI（Top6A）的催化亚基同源。在 Top6A 晶体结构的指导下，我们对 rec12 基因座进行了工程改造，使 Rec12 蛋白的每个保守残基都有一个单一的氨基酸取代。在 21 个取代中，有 10 个显著降低或消除了减数分裂 DSB、基因转换、交叉重组和染色体的正确分离。关键残基位于金属离子结合口袋内的 prim（E179A、D229A、D231A）、催化区域 5Y-CAP（R94A、D95A、Y98F）和 DNA 结合界面（K201A、G202E、R209A、K242A）。一组取代降低了 DSB，但保持了交叉，表明交叉保持平衡。此外，一个强分离功能突变（R304A）表明，交叉/非交叉决定是由 Rec12 的蛋白质-蛋白质相互作用表面在早期建立的。裂殖酵母每个二价体有多个交叉，在大约每个二价体一个交叉的阈值以上，染色体分离是稳健的，低于这个阈值，就会发生非分离。这些结果支持 Rec12/Spo11/Top6A 家族成员在 DSB 催化中的结构和功能保守性，并且它们揭示了 Rec12 如何调节减数分裂染色体动力学的其他特征。
