Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells

DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the α subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.

DNA解旋酶B（DNA helicase B）是小鼠FM3A细胞中的主要DNA解旋酶。从FM3A细胞中分离得到的温度敏感DNA复制缺陷突变株tsFT848，其DNA解旋酶B具有热不稳定性。本研究中，我们从小鼠FM3A细胞中纯化得到DNA解旋酶B，并测定了该纯化蛋白的部分氨基酸序列。利用根据其中一条部分氨基酸序列合成的DNA探针（DNA probe），我们分离得到一条互补DNA（cDNA），该序列编码一款分子量为121.5 kDa的蛋白质，且包含DNA/RNA解旋酶超家族成员共有的7个保守基序。数据库检索结果显示，DNA解旋酶B与包括大肠杆菌RecD蛋白在内的多种原核生物的脱氧核糖核酸外切酶V（exodeoxyribonuclease V）α亚基具有序列相似性，但在酵母中未发现其同源蛋白。我们对来自tsFT848的编码DNA解旋酶B的互补DNA进行了测序，发现在DNA/RNA解旋酶基序IV与V之间存在一处突变。