Title: Premature Structural Changes at Replication Origins in a Yeast Minichromosome Maintenance (MCM) Mutant
Abstract: The Cdc7p protein kinase in the budding yeast<i>Saccharomyces cerevisiae</i> is thought to help trigger DNA replication by modifying one or more of the factors that assemble at replication origins (ARSs). To investigate events catalyzed by Cdc7p, we compared the structure of replication origins in cells containing conditional mutations in Cdc7p and Cdc8p, a thymidylate kinase that is required for DNA synthesis. High resolution genomic footprinting indicated that the presumptive lagging strand template in ARS1 became highly sensitive to KMnO<sub>4</sub> modification after the<i>CDC7</i> execution point. These results suggested that Cdc7p triggers DNA unwinding. The transition from late G<sub>1</sub> phase to the <i>CDC7</i> execution point and from the <i>CDC7</i>to the <i>CDC8</i> execution points was accompanied by small but ARS-dependent changes in DNA topology. These results suggested that DNA unwinding before the <i>CDC8</i> execution point either is highly localized or that the torsional stress associated with initial DNA unwinding is minimized by compensatory protein-DNA structural changes. The ARS DNA structural attributes evident in cells blocked at the <i>CDC8</i> execution point were also evident in α-factor-blocked, G<sub>1</sub> phase cells containing the <i>CDC7</i> bypass mutant<i>mcm5/cdc46-bob1</i>. This result strongly suggests that the structural changes during the transition from the <i>CDC7</i> to<i>CDC8</i> execution points depend on the Cdc7p protein kinase and involve alteration of the minichromosome maintenance protein complex.