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Molecular and Cellular Biology, December 1999, p. 8003-8015, Vol. 19, No. 12
Mitsubishi Kasei Institute of Life Sciences,
Machida, Tokyo 194-8511, Japan
Received 19 March 1999/Returned for modification 30 April
1999/Accepted 15 August 1999
Mcm proteins play an essential role in eukaryotic DNA replication,
but their biochemical functions are poorly understood. Recently, we
reported that a DNA helicase activity is associated with an
Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is
involved in the initiation of DNA replication as a DNA-unwinding
enzyme. In this study, we have expressed and isolated the mouse
Mcm2,4,6,7 proteins from insect cells and characterized various mutant
Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and
Mcm6 proteins were mutated. The activities associated with such
preparations demonstrated that the DNA helicase activity is
intrinsically associated with the Mcm4,6,7 complex. Biochemical
analyses of these mutant Mcm4,6,7 complexes indicated that the ATP
binding activity of the Mcm6 protein in the complex is critical for DNA
helicase activity and that the Mcm4 protein may play a role in the
single-stranded DNA binding activity of the complex. The results also
indicated that the two activities of DNA helicase and single-stranded
DNA binding can be separated.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Biochemical Analysis of the Intrinsic
Mcm4-Mcm6-Mcm7 DNA Helicase Activity
*
Corresponding author. Mailing address: Mitsubishi Kasei
Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. Phone: 81-42-724-6266. Fax: 81-42-724-6314. E-mail:
yukio{at}libra.ls.m-kagaku.co.jp.
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