Huang R Y, Kowalski D
Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Nucleic Acids Res. 1996 Mar 1;24(5):816-23. doi: 10.1093/nar/24.5.816.
A yeast autonomously replicating sequence, ARS305, shares essential components with a chromosome III replicator, ORI305. Known components include an ARS consensus sequence (ACS) element, presumed to bind the origin recognition complex (ORC), and a broad 3'-flanking sequence which contains a DNA unwinding element. Here linker substitution mutagenesis of ARS305 and analysis of plasmid mitotic stability identified three short sequence elements within the broad 3'-flanking sequence. The major functional element resides directly 3' of the ACS and the two remaining elements reside further downstream, all within non-conserved ARS sequences. To determine the contribution of the elements to replication origin function in the chromosome, selected linker mutations were transplaced into the ORI305 locus and two-dimensional gel electrophoresis was used to analyze replication bubble formation and fork directions. Mutation of the major functional element identified in the plasmid mitotic stability assay inactivated replication origin function in the chromosome. Mutation of each of the two remaining elements diminished both plasmid ARS and chromosomal origin activities to similar levels. Thus multiple DNA elements identified in the plasmid ARS are determinants of replication origin function in the natural context of the chromosome. Comparison with two other genetically defined chromosomal replicators reveals a conservation of functional elements known to bind ORC, but no two replicators are identical in the arrangement of elements downstream of ORC binding elements or in the extent of functional sequences adjacent to the ACS.
酵母自主复制序列ARS305与III号染色体复制起点ORI305具有一些共同的基本元件。已知元件包括一个假定可结合起始识别复合体（ORC）的ARS共有序列（ACS）元件，以及一个包含DNA解旋元件的宽泛的3'侧翼序列。在此，我们对ARS305进行了接头置换诱变，并分析了质粒的有丝分裂稳定性，从而在宽泛的3'侧翼序列中确定了三个短序列元件。主要功能元件直接位于ACS的3'端，另外两个元件位于更下游，均在非保守的ARS序列内。为了确定这些元件对染色体复制起点功能的贡献，我们将选定的接头突变转移到ORI305位点，并使用二维凝胶电泳分析复制泡的形成和叉的方向。在质粒有丝分裂稳定性测定中鉴定出的主要功能元件发生突变后，会使染色体中的复制起点功能失活。另外两个元件中的每一个发生突变，都会使质粒ARS和染色体起点活性降低到相似水平。因此，在质粒ARS中鉴定出的多个DNA元件是染色体自然环境中复制起点功能的决定因素。与其他两个经过基因定义的染色体复制起点进行比较发现，已知可结合ORC的功能元件具有保守性，但在ORC结合元件下游的元件排列或与ACS相邻的功能序列范围方面，没有两个复制起点是相同的。
