Tsukamoto Y, Kato J, Ikeda H
Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Japan.
Mol Gen Genet. 1997 Aug;255(5):543-7. doi: 10.1007/s004380050527.
We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid. In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis. We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers. Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid. In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain. The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis.
我们之前已经表明，酿酒酵母的RAD50、RAD52、MRE11、XRS2和HDF1基因参与了单中心质粒上非法重组导致的缺失形成。在本研究中，我们研究了这些基因的突变对双中心质粒缺失形成的影响，在双中心质粒中，由于两个着丝粒在有丝分裂中被拉向相反的两极，预计DNA双链断裂会频繁发生。我们用双中心质粒转化酵母细胞，在经过几个分裂周期的培养后，使用负选择标记检测携带缺失质粒的细胞。与单中心质粒相比，缺失发生的频率更高，并且在重组连接处有短的同源区域，这与在单中心质粒上观察到的情况相同。在rad50、mre11、xrs2和hdf1突变体中，缺失发生的频率降低了约50倍，而在rad52突变体中，其与野生型菌株相当。Rad50、Mre11、Xrs2和Hdf1的末端连接功能表明，这些蛋白质在有丝分裂期间双中心质粒上产生的DNA末端连接中起重要作用。
