McIntosh E M, Ager D D, Gadsden M H, Haynes R H
Department of Biology, York University, Toronto, Ontario, Canada.
Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8020-4. doi: 10.1073/pnas.89.17.8020.
Two functional human dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) cDNAs were isolated from a cDNA expression library by genetic complementation in Escherichia coli. These cDNAs differed in size but exhibited a common overlapping DNA sequence. Contained within this sequence was a single long open reading frame sufficient to encode a polypeptide of 141 amino acids with a calculated molecular mass of 16.6 kDa. The amino acid sequence of this protein exhibits 35% identity with the E. coli dUTPase and 53% identity with the Saccharomyces cerevisiae enzyme. The human dUTPase was found to contain five characteristics amino acid sequence motifs that are common to the dUTPases of E. coli, yeast, and herpesviruses and to dUTPase-like sequences encoded by some retrovirus gag and pol genes. A high degree of amino acid sequence identity (greater than 60%) was also observed between the human dUTPase and the putative pseudoproteases of two poxviruses, indicating that these virus proteins are dUTPases. Northern hybridization analysis reveals that dUTPase is encoded by at least two species of poly(A)+ mRNA and possibly a third, smaller species. All of these mRNAs are present in a variety of human tissues but their relative levels vary between tissues. Southern analysis indicates that the dUTPase gene has been conserved to some extent throughout vertebrate evolution; however, the gene may be very large, or its organization somewhat complex in some systems. We suggest that dUTPase may generally perform an essential role in DNA replication and therefore could serve as a target enzyme for the development of chemotherapeutic compounds.
通过在大肠杆菌中的基因互补，从一个cDNA表达文库中分离出了两个功能性人源dUTP焦磷酸酶（dUTPase；EC 3.6.1.23）cDNA。这些cDNA大小不同，但呈现出一个共同的重叠DNA序列。该序列中包含一个单一的长开放阅读框，足以编码一个141个氨基酸的多肽，计算分子量为16.6 kDa。这种蛋白质的氨基酸序列与大肠杆菌dUTPase有35%的同一性，与酿酒酵母的酶有53%的同一性。发现人源dUTPase含有五个特征性氨基酸序列基序，这些基序是大肠杆菌、酵母和疱疹病毒的dUTPase以及一些逆转录病毒gag和pol基因编码的dUTPase样序列所共有的。在人源dUTPase与两种痘病毒的假定假蛋白酶之间也观察到高度的氨基酸序列同一性（大于60%），表明这些病毒蛋白是dUTPase。Northern杂交分析显示，dUTPase由至少两种多聚腺苷酸加尾（poly(A)+）mRNA编码，可能还有第三种较小的mRNA。所有这些mRNA都存在于多种人体组织中，但它们的相对水平在不同组织之间有所变化。Southern分析表明，dUTPase基因在整个脊椎动物进化过程中在一定程度上得到了保守；然而，该基因可能非常大，或者在某些系统中其结构有些复杂。我们认为，dUTPase可能通常在DNA复制中发挥重要作用，因此可作为开发化疗化合物的靶标酶。
