[Show abstract][Hide abstract] ABSTRACT: Proper eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) but not S phase cyclin-dependent kinase (S-CDK) is required for the initial origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Likewise, in vivo, DDK drives early-firing-origin recruitment of Cdc45 before activation of S-CDK. After S-CDK activation, a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Finally, recruitment of lagging but not leading strand DNA polymerases depends on Mcm10 and DNA unwinding. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to origin DNA unwinding and RNA primer synthesis.
[Show abstract][Hide abstract] ABSTRACT: In Saccharomyces cerevisiae cells, B-type cyclin-dependent kinases (CDKs) target two origin recognition complex (ORC) subunits, Orc2 and Orc6, to inhibit helicase loading. We show that helicase loading by ORC is inhibited by two distinct CDK-dependent mechanisms. Independent of phosphorylation, binding of CDK to an "RXL" cyclin-binding motif in Orc6 sterically reduces the initial recruitment of the Cdt1/Mcm2-7 complex to ORC. CDK phosphorylation of Orc2 and Orc6 inhibits the same step in helicase loading. This phosphorylation of Orc6 is stimulated by the RXL motif and mediates the bulk of the phosphorylation-dependent inhibition of helicase loading. Direct binding experiments show that CDK phosphorylation specifically blocks one of the two Cdt1-binding sites on Orc6. Consistent with the inactivation of one Cdt1-binding site preventing helicase loading, CDK phosphorylation of ORC causes a twofold reduction of initial Cdt1/Mcm2-7 recruitment but results in nearly complete inhibition of Mcm2-7 loading. Intriguingly, in addition to being a target of both CDK inhibitory mechanisms, the Orc6 RXL/cyclin-binding motif plays a positive role in the initial recruitment of Cdt1/Mcm2-7 to the origin, suggesting that this motif is critical for the switch between active and inhibited ORC function at the G1-to-S-phase transition.
Genes & development 02/2011; 25(4):363-72. DOI:10.1101/gad.2011511 · 10.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The origin recognition complex (ORC) nucleates DNA replication initiation in eukaryotic cells. This six-protein complex binds replication origin DNA, recruits other initiation factors, and facilitates loading of the DNA helicase. Studying the function of individual ORC subunits during pre-RC formation has been hampered by the requirement of most subunits for DNA binding. In this study, we investigate the function of the Saccharomyces cerevisiae Orc6, the only ORC subunit not required for DNA binding. In vivo, depletion of Orc6 inhibits prereplicative complex (pre-RC) assembly and maintenance. In vitro, ORC lacking Orc6 fails to interact with Cdt1 and to load the Mcm2-7 helicase onto origin DNA. We demonstrate that two regions of Orc6 bind Cdt1 directly, and that the extreme C terminus of Orc6 (Orc6-CTD) interacts tightly with the remaining five ORC subunits. Replacing Orc6 with a fusion protein linking Cdt1 to the Orc6-CTD results in an ORC complex that loads Mcm2-7 onto DNA. Interestingly, this complex can only perform a single round of Mcm2-7 loading, suggesting that a dynamic association of Cdt1 with ORC is required for multiple rounds of Mcm2-7 loading.
Genes & Development 12/2007; 21(22):2897-907. DOI:10.1101/gad.1596807 · 10.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The origin recognition complex (ORC) is a six-subunit, ATP-regulated, DNA binding protein that is required for the formation of the prereplicative complex (pre-RC), an essential replication intermediate formed at each origin of DNA replication. In this study, we investigate the mechanism of ORC function during pre-RC formation and how ATP influences this event. We demonstrate that ATP hydrolysis by ORC requires the coordinate function of the Orc1 and Orc4 subunits. Mutations that eliminate ORC ATP hydrolysis do not support cell viability and show defects in pre-RC formation. Pre-RC formation involves reiterative loading of the putative replicative helicase, Mcm2-7, at the origin. Importantly, preventing ORC ATP hydrolysis inhibits this repeated Mcm2-7 loading. Our findings indicate that ORC is part of a helicase-loading molecular machine that repeatedly assembles Mcm2-7 complexes onto origin DNA and suggest that the assembly of multiple Mcm2-7 complexes plays a critical role in origin function.