Biochemical Characterization Of Mcm4Chaos3 Mutation Using An Archaeal Mcm As A Model.
ABASTRACT The minichromosome maintenance (MCM) complex is essential for DNA replication in eukarya and archaea and is thought to function as the replicative helicase. While in eukarya the MCM is a complex of six highly conserved but nonidentical polypeptides (Mcm2-7), most archaeal species contain 6 identical polypeptides. In a N-ethyl-Nnitrosourea (ENU) mutagenesis screen for mice showing chromosome instability, a mutant mouse was identified which harbored a mutation in MCM4 protein referred to as Chaos3 (chromosome aberrations occurring spontaneously 3). MCM4Chaos3 mice harbor a single amino acid substitution (F345I) in which a conserved phenylalanine residue in MCM4 protein is replaced by isoleucine. Female mice homozygous for the mutation are susceptible to mammary adenocarcinomas. Similar mutations in the budding yeast homologue of MCM4 resulted in a strong minichromosome loss phenotype. Using the single MCM homologue from the archaeon Methanothermobacter thermautotrophicus (mtMCM), we show that the equivalent mutation (F172I) affects the stability and activity of the helicase. The implications of these observations for the eukaryotic MCM functions are discussed. Besides the biochemical characterization, I have carried out structural studies of mutant proteins using Small Angle X-ray Scattering (SAXS) to illustrate the structural effect of F172I mutation. Some preliminary results are presented to show that F172I mutation does affect the structural integrity of N-terminal mtMCM protein.
MCM4Chaos3 mutation; Archaeal MCM; MCM4; MCM complex; MCM2-7; hexamer structure; Helicase; ATPase; DNA binding
Gruner, Sol Michael
Tye, Bik-Kwoon; Cerione, Richard A
M.S. of Biophysics
Master of Science
dissertation or thesis