Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B

Masayuki Seki, Takeo Kohda, Tamaki Yano, Shusuke Tada, Junn Yanagisawa, Toshihiko Eki, Michio Ui, Takemi Enomoto

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA- dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39°C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39°C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

Original languageEnglish
Pages (from-to)165-172
Number of pages8
JournalMolecular and cellular biology
Volume15
Issue number1
DOIs
Publication statusPublished - 1995 Jan
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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