el-Hajj H H, Wang L, Weiss B
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109-0602.
J Bacteriol. 1992 Jul;174(13):4450-6. doi: 10.1128/jb.174.13.4450-4456.1992.
The dut gene of Escherichia coli encodes deoxyuridine triphosphatase, an enzyme that prevents the incorporation of dUTP into DNA and that is needed in the de novo biosynthesis of thymidylate. We produced a conditionally lethal dut(Ts) mutation and isolated a phenotypic revertant that had a mutation in an unknown gene tentatively designated dus (for dut suppressor). The dus mutation restored the ability of the dut mutant to grow at 42 degrees C without restoring its enzymatic activity or thymidylate independence. A strain was constructed bearing, in addition to these mutations, ones affecting the following genes and their corresponding products: ung, which produces uracil-DNA N-glycosylase, a repair enzyme that removes uracil from DNA; deoA, which produces thymidine (deoxyuridine) phosphorylase, which would degrade exogenous deoxyuridine; and thyA, which produces thymidylate synthase. When grown at 42 degrees C in minimal medium containing deoxyuridine, the multiple mutant displayed a 93 to 96% substitution of uracil for thymine in new DNA. Growth stopped after the cellular DNA had increased 1.6- to 1.9-fold and the cell mass had increased 1.7- to 2.7-fold, suggesting a general failure of macromolecular biosynthesis. DNA hybridization confirmed that the uracil-containing DNA was chromosomal and that new rounds of initiation must have occurred during its synthesis.
大肠杆菌的dut基因编码脱氧尿苷三磷酸酶，该酶可防止dUTP掺入DNA，是胸苷酸从头生物合成所必需的。我们产生了一个条件致死性的dut（Ts）突变，并分离出一个表型回复突变体，该突变体在一个暂定为dus（dut抑制子）的未知基因中发生了突变。dus突变恢复了dut突变体在42℃下生长的能力，但没有恢复其酶活性或胸苷酸自主性。构建了一个菌株，除了这些突变外，还带有影响以下基因及其相应产物的突变：ung，其产生尿嘧啶-DNA N-糖苷酶，一种从DNA中去除尿嘧啶的修复酶；deoA，其产生胸苷（脱氧尿苷）磷酸化酶，该酶会降解外源性脱氧尿苷；thyA，其产生胸苷酸合成酶。当在含有脱氧尿苷的基本培养基中于42℃下生长时，多重突变体在新合成的DNA中显示出93%至96%的尿嘧啶替代胸腺嘧啶。在细胞DNA增加1.6至1.9倍且细胞质量增加1.7至2.7倍后生长停止，这表明大分子生物合成普遍失败。DNA杂交证实含尿嘧啶的DNA是染色体DNA，并且在其合成过程中一定发生了新一轮的起始。
