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

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.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...

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

“…To analyze whether the C-tier precedes the NTD during translocation of CMG, we initially assembled CMG onto a synthetic DNA fork containing a 25-mer doublestranded DNA (dsDNA) stem, a leading ssDNA 3′ dT 40 tail, and a 16-mer 5′ (CCGA) 4 ssDNA tail. The (CCGA) 4 sequence was used for the lagging strand because CMG does not bind this sequence, whereas CMG binding to dT 40 is readily apparent (18). CMG was mixed with a 4.2-fold molar excess of DNA fork and 0.2 mM Adenylylimidodiphosphate (AMPPNP) followed by gel filtration to remove excess DNA (SI Appendix, Fig.…”

The architecture of a eukaryotic replisome

“…6). Indeed, a recent study of Drosophila CMG demonstrates the leading strand can occupy the second channel under particular conditions 37 . Alternatively, the leading ssDNA may take a ~20 nucleotide path by exiting the Mcm channel at an internal position (e.g.…”

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase

“…For example, the ssDNA generated by one or both of these events could interact with Cdc45 or GINS (Costa et al 2014), resulting in stabilized CMG complexes. Supporting this possibility, Cdc45 is related to the bacterial RecJ ssDNA nuclease and has been shown to bind ssDNA (Bruck and Kaplan 2013;Petojevic et al 2015). Finally, given the potential role of OB-fold domains in ssDNA interactions (Ashton et al 2013;Froelich et al 2014), it is also possible that release from the A subdomain allows the Mcm2 OB-fold domain to form more productive interactions with translocating ssDNA.…”