Structural and Functional Insights into the DNA Replication Factor Cdc45 Reveal an Evolutionary Relationship to the DHH Family of Phosphoesterases

Krastanova, I.; Sannino, Vincenzo; Amenitsch, Heinz; Gileadi, O.; Pisani, Francesca M.; Onesti, Silvia

doi:10.1074/jbc.m111.285395

Cited by 53 publications

(72 citation statements)

References 41 publications

(35 reference statements)

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“…We speculate here, and also in a report of a recent EM structure of the CMG complex (18), that Cdc45 may bind to the lagging strand that is sterically excluded from the replicative helicase. We also speculate that Cdc45-ssDNA binding may chaperone the lagging strand toward the polymerase (45). We find that our Cdc45 mutant defective in binding ssDNA is competent for DNA replication under normal conditions.…”

Section: Discussionmentioning

confidence: 63%

“…A recent report describes homology between RecJ and Cdc45, and the report also demonstrates that human Cdc45 can bind ssDNA (45). We speculate here, and also in a report of a recent EM structure of the CMG complex (18), that Cdc45 may bind to the lagging strand that is sterically excluded from the replicative helicase.…”

Section: Discussionmentioning

confidence: 75%

“…This region of Cdc45 is not homologous to RecJ (26). There is a very recent report that human Cdc45 binds to ssDNA in vitro (45). It will be interesting to determine in the future whether the mechanism we revealed for Cdc45 dissociation from Mcm2-7 in budding yeast is also operative in mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

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Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Bruck

Kaplan

2013

Journal of Biological Chemistry

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Background: Polymerase stalling is coupled with helicase stalling at a eukaryotic replication fork by an unknown mechanism. Results: When a Cdc45-ssDNA binding mutant is expressed in budding yeast cells exposed to hydroxyurea, there is uncoupling of the helicase from the polymerase. Conclusion: Cdc45-ssDNA interaction is important during replication stress. Significance: A new model explains how polymerase stalling is coupled with helicase stalling.

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Section: Discussionmentioning

confidence: 63%

Section: Discussionmentioning

confidence: 75%

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Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Bruck

Kaplan

2013

Journal of Biological Chemistry

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“…Collectively, these data suggest that the gate between Mcm2 and Mcm5 can open at given points and that on these occasions the leading strand may dissociate from the central channel; in such instances, the side channel formed by GINS and Cdc45 would help prevent CMG dissociation and enable reestablishment of productive translocation. Together, our data underscore a potential role for Cdc45 in maintaining contacts with both the leading and lagging strands when the helicase may be stalled-such as during S-phase stress-and where the open gate conformation may persist, a point emphasized by the homology of Cdc45 to the prokaryotic repair protein RecJ (21,26).…”

mentioning

confidence: 91%

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

Petojevic

Pesavento

Costa³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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.hromosomal DNA replication begins with the separation of the complementary strands of the duplex. Following this melting step, helicases continuously separate the paired strands, exposing the template for enzymatic synthesis. For eukaryotic DNA replication, a growing body of work suggests that the initial DNA melting step involves an enzymatic conversion of a double hexamer of the minichromosome maintenance (Mcm2-7) complex into an active helicase, with hexamer separation forming two forks moving in opposite directions (1). The molecular mechanisms and a complete list of the factors that work to achieve melting and the topological conversion of double to single strand are still unknown, but both Cdc45 and the GINS complex are present at the time of melting (2) and are critical components of an activated Mcm2-7 helicase (3-6). Understanding the initiation process requires studies focused on the various transitions accessed by Mcm2-7 proteins and defining what roles the GINS and Cdc45 may play in the melting and unwinding processes.The CMG helicase characterized in vitro in Drosophila and humans contains a single Cdc45 protein, a single hetero-hexameric Mcm2-7, and a tetrameric GINS complex (3-7). Mcm2-7 acts as the motor that drives helicase activity; however, for metazoans, only when the Mcm2-7 complex is associated with Cdc45 and GINS do significant helicase, ATPase, and DNA binding activities follow (4). The two Mcm2-7 complexes are first loaded to DNA by mechanisms that appear to retain certain parallels to the processes used for loading sliding processivity clamps onto DNA. The endpoint of loading results in the deposition of a duplex DNA that runs through the central channel of an MCM double hexamer. Although to...

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“…Similar to the CMG, the GINS tetramer can bind to DNA substrates containing single-stranded regions but not to bluntended duplexes (193,224). Cdc45, which appears to be a catalytically inactive homolog of the bacterial/archaeal 5′→3′ RecJ exonuclease (225), also associates with ssDNA but not ds-DNA (226). Within the CMG, two topologically segregated conduits exist, one formed by the Mcm2-7 ring and the other between the external face of Mcm2 · 5 · 3 and one side of GINS · Cdc45 (Figure 5e) (170).…”

Section: Helicase Loading and Activation In Eukaryotesmentioning

confidence: 99%

Mechanisms for initiating cellular DNA replication

Bleichert

Botchan

Berger

2017

Science

189

172

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The initiation of DNA replication represents a committing step to cell proliferation. Appropriate replication onset depends on multiprotein complexes that help properly distinguish origin regions, generate nascent replication bubbles, and promote replisome formation. This review describes initiation systems employed by bacteria, archaea, and eukaryotes, with a focus on comparing and contrasting molecular mechanisms among organisms. Although commonalities can be found in the functional domains and strategies used to carry out and regulate initiation, many key participants have markedly different activities and appear to have evolved convergently. Despite significant advances in the field, major questions still persist in understanding how initiation programs are executed at the molecular level.

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Structural and Functional Insights into the DNA Replication Factor Cdc45 Reveal an Evolutionary Relationship to the DHH Family of Phosphoesterases

Cited by 53 publications

References 41 publications

Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

Mechanisms for initiating cellular DNA replication

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