Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe
Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe
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DOI：
10.1073/pnas.93.1.502
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发表时间：
1996-01-09
影响因子：
11.1
通讯作者：
Hurwitz, J
中科院分区：
文献类型：
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作者：
Murakami, Y;Huberman, JA;Hurwitz, J
裂殖酵母的自主复制序列(ARS)元件含有Maundrell等人报道的共有序列的多个不完整拷贝。[Maundrell K，Hutchison，A，&Son，S(1988)EMBO J.7,2203-2209]。当S、蓬布的无细胞提取物与含有ARS共有序列的寡核苷酸的二聚体或四聚体孵育时，凝胶迁移率漂移分析检测到几个复合体。形成这些复合体的蛋白质还与ars3002结合，ars3002是庞贝S第三染色体ura4区域最活跃的起源。其中一个蛋白被纯化为60 kDa的蛋白，命名为ARS结合蛋白1(Abp1)。Abp1优先结合到ARS 3002中的多个位点和DNA聚合物Poly[d(A。T)]。Abp1基因的克隆和序列分析表明，Abp1编码59.8 kDa的蛋白质(522个氨基酸)，Abp1与人和小鼠着丝粒DNA结合蛋白CENP-B的N末端有显著的同源性(25%的同源性，50%的相似性)(约300个氨基酸)。由于S.pombe的着丝粒含有高密度的ARS元件，Abp1可能在DNA复制和染色体分离中发挥作用。
Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K,, Hutchison, A, & Shall, S, (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S, pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S, pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity, It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A . T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids), Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximate to 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.