Cdc45-ssDNA interaction is important for stalling the helicase during replication stress.
ABSTRACT Replicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly to long (>40 nucleotides) genomic single-stranded DNA (ssDNA), and 60mer ssDNA specifically disrupts the interaction between Cdc45 and Mcm2-7. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is expressed in budding yeast cells exposed to hydroxyurea, cell growth is severely inhibited and excess RPA accumulates at or near an origin. Chromatin immunoprecipitation suggests that helicase movement is uncoupled from polymerase movement for mutant cells exposed to hydroxyurea. These data suggest that Cdc45-ssDNA interaction is important for stalling the helicase during replication stress.
SourceAvailable from: Jingdan Liang[Show abstract] [Hide abstract]
ABSTRACT: DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2-7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2-7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2-7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel.Proceedings of the National Academy of Sciences 01/2015; DOI:10.1073/pnas.1422003112 · 9.81 Impact Factor
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ABSTRACT: Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-CDK phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7 and GINS. We show here using purified proteins from budding yeast that Dpb11 alone binds to Mcm2-7, and Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased RPA interaction with origin DNA during S phase. We propose a model wherein Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, while bound to Cdc45-Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon the extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7 and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45-Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 02/2015; 290(12). DOI:10.1074/jbc.M115.640383 · 4.60 Impact Factor
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ABSTRACT: The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS (CMG). The Dbf4-Cdc7 kinase phosphorylates Mcm2 in vitro, but the in vivo role for Dbf4-Cdc7 phosphorylation of Mcm2 is unclear. We find that budding yeast Dbf4-Cdc7 phosphorylates Mcm2 in vivo under normal conditions during S phase. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 confers a dominant-negative phenotype with a severe growth defect. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 under wild-type-expression conditions also results in impaired DNA replication, substantially decreased single-stranded formation at an origin, and markedly disrupted interaction between GINS and Mcm2-7 during S phase. In vitro, DDK phosphorylation of Mcm2 substantially weakens the interaction between Mcm2 and Mcm5, and Dbf4-Cdc7 phosphorylation of Mcm2 promotes Mcm2-7 ring opening. The extrusion of ssDNA from the central channel of Mcm2-7 triggers GINS attachment to Mcm2-7. Thus, Dbf4-Cdc7 phosphorylation of Mcm2 may open the Mcm2-7 ring at the Mcm2-Mcm5 interface, allowing for single-stranded DNA extrusion and subsequent GINS assembly with Mcm2-7. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 12/2014; 290(2). DOI:10.1074/jbc.M114.608232 · 4.60 Impact Factor