Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in Saccharomyces cerevisiae.
ABSTRACT Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G(1) phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.
The Israel Medical Association journal: IMAJ 01/2003; 4(12):1135-7. · 1.02 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The product of the CDC7 gene of Saccharomyces cerevisiae is known to be required in the mitotic cell cycle for the initiation of DNA replication. We show that changes in transcript levels do not account for this stage-specific function, since the steady-state mRNA concentration remains constant at 1 copy per cell throughout the cell cycle. By measuring the cell division capacity of a cdc7::URA3 mutant after loss of a single-copy plasmid containing the CDC7 gene, we show that the CDC7 protein is present in at least 200-fold excess of the amount required for a single cell division. These results appear to exclude periodic transcription or translation as a means by which CDC7 function is regulated. In contrast, the CDC7 protein is known to be dispensable for meiotic S phase, but is required for synaptonemal complex formation and recombination. We found that the CDC7 transcript level does vary during meiosis, reaching a maximum near the time at which recombination occurs. Meiotic spores containing a cdc7 null allele germinate but fail to complete cell division. Apparently the excess CDC7 product present in mitotic cells is physically excluded from the spores (or becomes inactivated) and must be produced de novo after germination. The cdc7-1 allele had previously been shown to confer a reduction in the rate of induced mutation. We show that the cloned wild-type CDC7 gene not only complements this defect, but that when the CDC7 gene is on a multiple copy plasmid, induced mutagenesis is increased. Therefore, in contrast to the excess CDC7 activity for cell division, the level of activity for some error-prone repair process may be normally limiting.Molecular and Cellular Biology 02/1988; 8(1):293-300. · 5.53 Impact Factor
Article: [Stress echocardiography versus myocardial scintigraphy: comparative value in coronary heart disease].Herz 05/1996; 21(2):136-41. · 0.92 Impact Factor