Primer utilization by DNA polymerase alpha-primase is influenced by its interaction with Mcm10p.
Models of DNA replication in yeast and Xenopus suggest that Mcm10p is required to generate the pre-initiation complex as well as progression of the replication fork during the elongation of DNA chains. In this report, we show that the Schizosaccharomyces pombe Mcm10p/Cdc23p binds to the S. pombe DNA polymerase (pol) alpha-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol alpha-primase complex with various primed DNA templates. We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol alpha-primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p. Their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA and to pol alpha were compared. Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA. Truncated derivatives of Mcm10p containing amino acids 1-416 retained the pol alpha binding activity, whereas the C terminus, amino acids 496-593, did not. These results demonstrate that both the single-stranded DNA binding and the pol alpha binding properties of Mcm10p play important roles in the activation. In accord with these findings, Mcm10p facilitated the binding of pol alpha-primase complex to primed DNA and formed a stable complex with pol alpha-primase on primed templates. A mutant that failed to activate or bind to DNA and pol alpha, was not observed in this complex. We suggest that the interaction of Mcm10p with the pol alpha-primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication.
Fien
Karen
K
Program in Molecular Biology, Memorial-Sloan Kettering Cancer Center, New York, New York 10021, USA.
Cho
Young-Sik
YS
Lee
Joon-Kyu
JK
Raychaudhuri
Santanu
S
Tappin
Inger
I
Hurwitz
Jerard
J
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D064110:Minichromosome Maintenance Proteins; D011485:Protein Binding; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D029702:Schizosaccharomyces pombe Proteins; C489609:cdc23 protein, S pombe; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I; D064110:Minichromosome Maintenance Proteins