Umbricht C B, Erdile L F, Jabs E W, Kelly T J
Department of Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1993 Mar 25;268(9):6131-8.
Replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, recombination, and repair. We have previously reported the cloning and bacterial expression of the 70- and 32-kDa subunits of human RPA (hRPA). We have now cloned the 14-kDa subunit (hRPA3) from a HeLa cell cDNA library. The hRPA3 cDNA is a 692-base pair sequence that contains an open reading frame encoding a protein of 121 amino acids with a calculated molecular mass of 13.6 kDa. The deduced amino acid sequence shows only limited similarity to the small subunit of yeast RPA and is unrelated to any other protein in the current data banks. A recombinant protein containing a short histidine tag at the NH2 terminus has been purified in good yield from Escherichia coli by metal-chelate affinity chromatography. Antibodies prepared against recombinant hRPA3 recognize the native protein and inhibit SV40 DNA replication in vitro. We have localized the genes for the 70-, 32-, and 14-kDa subunits to chromosomes 17, 1, and 7, respectively, using polymerase chain reaction amplification of genomic DNA from rodent-human hybrid cell lines. Since RPA appears to be involved in several fundamental cellular processes, the physical mapping of the RPA genes may be useful in identifying possible human genetic defects associated with RPA deficiency or dysfunction.
复制蛋白A（RPA）是一种由三个亚基组成的蛋白质，在真核生物DNA复制、重组及修复过程中发挥核心作用。我们之前已报道了人类RPA（hRPA）70 kDa和32 kDa亚基的克隆及细菌表达情况。现在我们从HeLa细胞cDNA文库中克隆出了14 kDa亚基（hRPA3）。hRPA3 cDNA是一段692个碱基对的序列，包含一个开放阅读框，编码一个由121个氨基酸组成的蛋白质，计算分子量为13.6 kDa。推导的氨基酸序列与酵母RPA的小亚基仅有有限的相似性，且与当前数据库中的任何其他蛋白质均无关联。一种在NH2末端带有短组氨酸标签的重组蛋白已通过金属螯合亲和层析从大肠杆菌中以高产率纯化出来。针对重组hRPA3制备的抗体可识别天然蛋白，并在体外抑制SV40 DNA复制。我们利用聚合酶链反应扩增啮齿动物 - 人类杂交细胞系的基因组DNA，将70 kDa、32 kDa和14 kDa亚基的基因分别定位到了17号、1号和7号染色体上。由于RPA似乎参与了多个基本细胞过程，RPA基因的物理图谱绘制可能有助于识别与RPA缺乏或功能障碍相关的潜在人类遗传缺陷。
