Biochemical Analysis of the Intrinsic Mcm4-Mcm6-Mcm7 DNA Helicase Activity

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Molecular and Cellular Biology, December 1999, p. 8003-8015, Vol. 19, No. 12
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

Zhiying You, Yuki Komamura, and Yukio Ishimi^*

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 associatedwith an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that thiscomplex is involved in the initiation of DNA replication as aDNA-unwinding enzyme. In this study, we have expressed and isolatedthe mouse Mcm2,4,6,7 proteins from insect cells and characterizedvarious mutant Mcm4,6,7 complexes in which the conserved ATPasemotifs of the Mcm4 and Mcm6 proteins were mutated. The activitiesassociated with such preparations demonstrated that the DNA helicaseactivity is intrinsically associated with the Mcm4,6,7 complex.Biochemical analyses of these mutant Mcm4,6,7 complexes indicatedthat the ATP binding activity of the Mcm6 protein in the complexis critical for DNA helicase activity and that the Mcm4 proteinmay play a role in the single-stranded DNA binding activity ofthe complex. The results also indicated that the two activitiesof DNA helicase and single-stranded DNA binding can beseparated.

^* 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.

Molecular and Cellular Biology, December 1999, p. 8003-8015, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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