Biochemical characterization of the Methanothermobacter thermautotrophicus minichromosome maintenance (MCM) helicase N-terminal domains.
Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases. For the archaeal protein it was shown that the N-terminal portion of the protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity. Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described. Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for protein multimerization and that domain B is the main contact region with single-stranded DNA. It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.
Kasiviswanathan
Rajesh
R
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA.
Shin
Jae-Ho
JH
Melamud
Eugene
E
Kelman
Zvi
Z
D000251:Adenosine Triphosphatases / Q000737:chemistry; D019843:Archaeal Proteins / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004265:DNA Helicases / Q000737:chemistry*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D017013:Methanobacteriaceae / Q000235:genetics*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012984:Software; D019809:Streptavidin / Q000737:chemistry; D020798:Two-Hybrid System Techniques
D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D019809:Streptavidin; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C403162:MCM protein, Methanobacterium thermoautotrophicum; D005990:Glycerol