Abstract
Assembly of replication complexes at the replication origins is strictly regulated. Cdc45p is known to be a part of the active replication complexes. In Xenopus egg extracts, Cdc45p was shown to be required for loading of DNA polymerase alpha onto chromatin. The fission yeast cdc45 homologue was identified as a suppressor for nda4 and named sna41. Nevertheless, it is not known how Cdc45p facilitates loading of DNA polymerase alpha onto chromatin, particularly to prereplicative complexes. To gain novel insight into the function of this protein in fission yeast, we characterized the fission yeast Cdc45 homologue, Sna41p. We have constructed C-terminally epitope-tagged Sna41p and Pol alpha p and replaced the endogenous genes with the corresponding tagged genes. Analyses of protein-protein interactions in vivo by the use of these tagged strains revealed the following: Sna41p interacts with Pol alpha p throughout the cell cycle, whereas it interacts with Mis5p/Mcm6p in the chromatin fractions at the G(1)-S boundary through S phase. In an initiation-defective sna41 mutant, sna41(goa1), interaction of Pol alpha p with Mis5p is not observed, although Pol alpha p loading onto the chromatin that occurs before G(1) START is not affected. These results show that fission yeast Sna41p facilitates the loading of Pol alpha p onto minichromosome maintenance proteins. Our results are consistent with a model in which loading of Pol alpha p onto replication origins occurs through two steps, namely, loading onto chromatin at preSTART and association with prereplicative complexes at G(1)-S through Sna41p, which interacts with minichromosome maintenance proteins in a cell cycle-dependent manner.
Highlights
Chromosomal DNA replication requires a series of complex events including recognition of origins, firing of replication origins, loading of DNA polymerases onto origins, and elongation of newly synthesized DNA
This association is followed by the loading of minichromosome maintenance (MCM)1 protein complexes onto the replication origins, leading to the formation of prereplicative complexes [6]
The arresting point of sna41goa1 is close to that of cdc20 or cdc22, and interaction of Sna41HA3p with MCMs takes place mainly at this cell cycle point. Taking these facts into account, we hypothesized that Sna41p is not required for chromatin loading of Pol␣p but is still required for association of Pol␣p with replicative complexes at the origins of DNA replication
Summary
All molecular biology techniques were performed as described previously [45]. Transformation of yeast strains was performed essentially by the lithium acetate procedure [33]. Three HA epitopes, linked by a single glycine, were inserted just before the termination codon of the sna open reading frame by consecutive PCR method. The PCR fragment was directly introduced into sna41⌬ strain rescued by pREP3XSna. The transformed cells were plated on yeast extract with supplements containing 5Ј-fluoroacetic acid. The 5Ј-fluoroacetic acid-resistant colonies, which could grow without pREP3X-Sna plasmid, were tested by PCR, genetic cross, and Western blot. Tandem FLAG tags, linked by a single glycine, were introduced before the termination codon of the pol␣ open reading frame as described previously [18], except that pUC18 carrying a ura nutritional marker instead of a leu marker was used
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