Kironmai K M, Muniyappa K, Friedman D B, Hollingsworth N M, Byers B
Department of Biochemistry, Indian Institute of Science, Bangalore.
Mol Cell Biol. 1998 Mar;18(3):1424-35. doi: 10.1128/MCB.18.3.1424.
The meiosis-specific HOP1 gene is important both for crossing over between homologs and for production of viable spores. hop1 diploids fail to assemble synaptonemal complex (SC), which normally provides the framework for meiotic synapsis. Immunochemical methods have shown that the 70-kDa HOP1 product is a component of the SC. To assess its molecular function, we have purified Hop1 protein to homogeneity and shown that it forms dimers and higher oligomers in solution. Consistent with the zinc-finger motif in its sequence, the purified protein contained about 1 mol equivalent of zinc whereas mutant protein lacking a conserved cysteine within this motif did not. Electrophoretic gel mobility shift assays with different forms of M13 DNA showed that Hop1 binds more readily to linear duplex DNA and negatively superhelical DNA than to nicked circular duplex DNA and even more weakly to single-stranded DNA. Linear duplex DNA binding was enhanced by the addition of Zn2+, was stronger for longer DNA fragments, and was saturable to about 55 bp/protein monomer. Competitive inhibition of this binding by added oligonucleotides suggests preferential affinity for G-rich sequences and weaker binding to poly(dA-dT). Nuclear extracts of meiotic cells caused exonucleolytic degradation of linear duplex DNA if the extracts were prepared from hop1 mutants; addition of purified Hop1 conferred protection against this degradation. These findings suggest that Hop1 acts in meiotic synapsis by binding to sites of double-strand break formation and helping to mediate their processing in the pathway to meiotic recombination.
减数分裂特异性HOP1基因对于同源染色体之间的交叉互换以及产生可育孢子都很重要。hop1二倍体无法组装联会复合体（SC），而联会复合体通常为减数分裂联会提供框架。免疫化学方法表明，70 kDa的HOP1产物是联会复合体的一个组成部分。为了评估其分子功能，我们已将Hop1蛋白纯化至同质，并表明它在溶液中形成二聚体和更高阶的寡聚体。与其序列中的锌指基序一致，纯化后的蛋白含有约1摩尔当量的锌，而在该基序内缺少保守半胱氨酸的突变蛋白则没有。用不同形式的M13 DNA进行的电泳凝胶迁移率变动分析表明，Hop1与线性双链DNA和负超螺旋DNA的结合比与带切口的环状双链DNA更容易，与单链DNA的结合则更弱。添加Zn2+可增强线性双链DNA的结合，较长的DNA片段结合更强，并且可饱和至约55 bp/蛋白单体。添加的寡核苷酸对这种结合的竞争性抑制表明对富含G的序列具有优先亲和力，而与聚（dA-dT）的结合较弱。如果从hop1突变体中制备减数分裂细胞的核提取物，则会导致线性双链DNA的核酸外切酶降解；添加纯化的Hop1可防止这种降解。这些发现表明，Hop1通过结合双链断裂形成位点并帮助介导其在减数分裂重组途径中的加工来参与减数分裂联会。
