Fission yeast Mcm10p contains primase activity.
Although Mcm10p is a conserved essential component in eukaryotes required for both the initiation and elongation of DNA chains, its biochemical properties are unknown. Here, we report that the Schizosaccharomyces pombe fission yeast Mcm10 protein contains primase activity. Primases are enzymes that synthesize RNA primers on single-stranded DNA templates that are extended by DNA polymerases. In keeping with this property, Mcm10p supported oligoribonucleotide synthesis of short RNA primers (preferentially initiating synthesis on a dT template) that were extended with dATP by Escherichia coli DNA polymerase I. The C terminus of Mcm10p synthesized RNA, but less efficiently than the full-length protein at low rNTP levels. Mcm10p homologs contain a C-terminal motif found in proteins that polymerize nucleotides. A point mutant within this motif of S. pombe Mcm10p was defective in primer synthesis in vitro, and this mutant failed to support growth in vivo, suggesting that the primase activity of Mcm10p may be essential for cell viability.
Fien
Karen
K
Program of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021. Electronic address: fienk@mskcc.org.
Hurwitz
Jerard
J
Program of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021. Electronic address: j-hurwitz@ski.mskcc.org.
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D019915:DNA Primase / Q000378:metabolism*; D007700:Kinetics; D064110:Minichromosome Maintenance Proteins; D009711:Nucleotides / Q000378:metabolism; D009843:Oligoribonucleotides / Q000096:biosynthesis; D010446:Peptide Fragments / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D013698:Templates, Genetic
D018797:Cell Cycle Proteins; D009711:Nucleotides; D009843:Oligoribonucleotides; D010446:Peptide Fragments; C034477:RNA primers; D029702:Schizosaccharomyces pombe Proteins; C489609:cdc23 protein, S pombe; D012313:RNA; D019915:DNA Primase; D064110:Minichromosome Maintenance Proteins