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

Abstract

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 alpha 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.

Figure 1

Purification of DNA helicase B. (A) The elution profile of ATPase and DNA helicase activities on the second Mono Q column. Open and closed circles represent ATPase activity in 5 µl of second Mono Q fractions with and without 5 µg of heat-denatured DNA, respectively. Bars in the graph indicate DNA helicase activity in 0.5 µl of the fractions. ³²P-labeled 21mer oligodeoxyribonucleotides liberated from M13mp19 single-stranded circular DNA were separated in a 12% polyacrylamide gel. The radioactivity of the liberated oligodeoxyribonucleotides was measured by BAS2000 (Fuji Photo Film) and plotted. A broken line indicates the KCl concentration calculated from the conductivity of the fractions. (B) SDS–PAGE of the Mono Q fractions. The eluted fractions (8 µl) were loaded onto a 7.5% SDS–polyacrylamide gel, and proteins were stained with Coomasie brilliant blue R-250. The arrowhead on the left of the gel indicates the position of the 130 kDa protein. Positions of molecular weight markers are indicated on the right.

Figure 2

Amino acid sequence of mouse DNA helicase B. The sequence was deduced from the longest open reading frame of a cDNA isolated from a fetal mouse brain cDNA library. The amino acid sequence of mouse DNA helicase B (HelB) was aligned with that of E.coli RecD protein (RecD) and B.subtilis YrrC protein (YrrC). Identical amino acids are boxed by black squares and similar amino acids are shaded. Partial amino acid sequences determined by using purified protein are indicated above the mouse DNA helicase B sequence by dots. The seven conserved DNA/RNA helicase motifs of mouse DNA helicase B, E.coli RecD protein and B.subtilis YrrC protein are underlined.

Figure 3

Determination of the mutation site of DNA helicase B in tsFT848 cells. (A) Schematic representation of RT–PCR for mapping of the mutation site in tsFT848 DNA helicase. The whole box represents the isolated DNA helicase B cDNA. An open reading frame and regions encoding seven conserved DNA/RNA helicase motifs are shown as shaded and black boxes, respectively. Primers for RT–PCR are indicated as arrows. The arrowhead indicates the position of the mutated nucleotide in tsFT848 DNA helicase B. (B) Analysis of DNA sequences of DNA helicase B cDNA from wild-type and tsFT848 cells. Charts from an automatic DNA sequencer corresponding to amino acids 723–725 of DNA helicase B are shown. Amino acid residues encoded by the nucleotide sequences are indicated on the top of the charts. Amino acid 724 in tsFT848 DNA helicase B is divergent from the wild-type residue and indicated in red.