Greenfeder S A, Newlon C S
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark 07103.
Mol Cell Biol. 1992 Sep;12(9):4056-66. doi: 10.1128/mcb.12.9.4056-4066.1992.
The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used two-dimensional agarose gel electrophoresis to analyze the replication of the chromosomal copies of yeast CEN1, CEN3, and CEN4 and determine the fate of replication forks that encounter the protein-DNA complex at the centromere. We have shown that replication fork pause sites are coincident with each of these centromeres and therefore probably with all yeast centromeres. We have analyzed the replication of plasmids containing mutant derivatives of CEN3 to determine whether the replication fork pause site is a result of an unusual structure adopted by centromere DNA or a result of the protein-DNA complex formed at the centromere. The mutant centromere derivatives varied in function as well as the ability to form the nuclease-resistant core structure. The data obtained from analysis of these derivatives indicate that the ability to cause replication forks to pause correlates with the ability to form the nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. Our findings further suggest that the centromere protein-DNA complex is present during S phase when replication forks encounter the centromere and therefore may be present throughout the cell cycle.
酵母着丝粒DNA的120个碱基对与蛋白质紧密结合，形成大小为200至240个碱基对的抗核酸酶核心结构。我们利用二维琼脂糖凝胶电泳分析了酵母CEN1、CEN3和CEN4染色体拷贝的复制情况，并确定了在着丝粒处遇到蛋白质-DNA复合物的复制叉的命运。我们已经表明，复制叉暂停位点与这些着丝粒中的每一个都一致，因此可能与所有酵母着丝粒一致。我们分析了含有CEN3突变衍生物的质粒的复制情况，以确定复制叉暂停位点是着丝粒DNA采用异常结构的结果，还是着丝粒处形成的蛋白质-DNA复合物的结果。突变着丝粒衍生物在功能以及形成抗核酸酶核心结构的能力方面各不相同。对这些衍生物的分析所获得的数据表明，导致复制叉暂停的能力与形成抗核酸酶核心结构的能力相关，而与特定DNA序列的存在与否无关。我们的研究结果进一步表明，当复制叉遇到着丝粒时，着丝粒蛋白质-DNA复合物在S期存在，因此可能在整个细胞周期中都存在。
