Abstract
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.
Highlights
In eukaryotes, the Mcm proteins are essential replication factors that were identified as proteins required for minichromosomal maintenance in a genetic screen for mutants defective in initiation of replication [2]
We demonstrate that full-length SpMcm10p and its C-terminal fragment contain primase activity that catalyzes the synthesis of oligoribonucleotides that are extended by Escherichia coli pol I
Mcm10p Catalyzes Template-dependent Synthesis of Oligoribonucleotides—We examined whether SpMcm10p and its Nand C-terminally truncated derivatives, Mcm10p-(1–303) and Mcm10p (416 –597), catalyze template-dependent oligoribonucleotide synthesis
Summary
The Mcm proteins are essential replication factors that were identified as proteins required for minichromosomal maintenance in a genetic screen for mutants defective in initiation of replication [2]. We demonstrate that full-length SpMcm10p (amino acids 1–593) and its C-terminal fragment (amino acids 416 –593) contain primase activity that catalyzes the synthesis of oligoribonucleotides that are extended by Escherichia coli pol I.
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