title: The PS1 hairpin of Mcm3 is essential for viability and for DNA unwinding in vitro.
abstract: The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3(K499A)) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3(K499A) strain. The ATPase activity of Mcm2-7(3K499A) was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Simon K W
authors.initials: SK
authors.lastname: Lam
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Ma
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Tina L
authors.initials: TL
authors.lastname: Sing
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Brian H
authors.initials: BH
authors.lastname: Shilton
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Brandl
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Megan J
authors.initials: MJ
authors.lastname: Davey
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003433:Crosses, Genetic; D004271:DNA, Fungal / Q000378:metabolism*; D050296:Microbial Viability / Q000187:drug effects; D064126:Minichromosome Maintenance Complex Component 3 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009153:Mutagens / Q000633:toxicity; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D010641:Phenotype; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D004271:DNA, Fungal; D009153:Mutagens; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C507761:MCM3 protein, S cerevisiae; D064126:Minichromosome Maintenance Complex Component 3