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PMID: 10567526 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Biochemical analysis of the intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity.

Molecular and cellular biology ·Vol. 19 ·No. 12 ·1999-12-00 ·Pages 8003-15

You Z, Komamura Y, Ishimi Y

Abstract

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.

MeSH Terms
Adenosine Triphosphatases/metabolism Animals Cell Cycle Proteins/genetics,isolation & purification,metabolism Chromosomal Proteins, Non-Histone DNA Helicases/genetics,isolation & purification,metabolism DNA, Single-Stranded/metabolism DNA-Binding Proteins/genetics,isolation & purification,metabolism Gene Expression Mice Minichromosome Maintenance Complex Component 2 Minichromosome Maintenance Complex Component 4 Minichromosome Maintenance Complex Component 6 Minichromosome Maintenance Complex Component 7 Mutagenesis Nuclear Proteins/genetics,isolation & purification,metabolism Protein Structure, Tertiary Saccharomyces cerevisiae Proteins
Chemicals
Cell Cycle Proteins Chromosomal Proteins, Non-Histone DNA, Single-Stranded DNA-Binding Proteins Nuclear Proteins Saccharomyces cerevisiae Proteins Adenosine Triphosphatases DNA Helicases MCM4 protein, S cerevisiae MCM6 protein, S cerevisiae MCM7 protein, S cerevisiae Mcm6 protein, mouse Mcm7 protein, mouse Minichromosome Maintenance Complex Component 2 Minichromosome Maintenance Complex Component 4 Minichromosome Maintenance Complex Component 6 Minichromosome Maintenance Complex Component 7
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
You Z
Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511, Japan.
Komamura Y
Ishimi Y
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.19.12.8003
Published
1999-12-00
Pages
8003-15
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC84885
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.19.12.8003 PMC Full Text View journal details
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