A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2–7 onto DNA

Samel, Stefan A.; Fernández-Cid, Alejandra; Sun, Jingchuan; Riera, Alberto; Tognetti, Silvia; Herrera, Mariano; H, Li; Speck, Christian

doi:10.1101/gad.242404.114

Cited by 114 publications

(113 citation statements)

References 55 publications

Supporting

Mentioning

108

Contrasting

Order By: Relevance

“…5D). Thus, our data show that Mcm2-7 loading must occur prior to double-hexamer formation, consistent with the finding that a single Mcm2-7 hexamer is loaded onto DNA during OCCM formation, although the first hexamer requires ORC-Cdc6 for stability (Sun et al 2013;Samel et al 2014). The Mcm2-7 double hexamer encircles dsDNA (Evrin et al 2009; (Fig.…”

Section: Function Of the Mcm2/5 Gate In The Mcm2-7 Double Hexamersupporting

confidence: 75%

“…Indeed, a DNA entry gate between Mcm2 and Mcm5 has been identified that is essential for pre-RC formation Samel et al 2014). If Mcm2-7 double-hexamer loading is a concerted process, as suggested Yardimci and Walter 2014), then DNA insertion into both Mcm2-7 hexamers needs to be coordinated, requiring the Mcm2/5 gate of each hexamer to be aligned to promote simultaneous ring opening.…”

Section: Function Of the Mcm2/5 Gate In The Mcm2-7 Double Hexamermentioning

confidence: 99%

See 1 more Smart Citation

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

et al. 2014

Self Cite

View full text Add to dashboard Cite

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

show abstract

Section: Function Of the Mcm2/5 Gate In The Mcm2-7 Double Hexamersupporting

confidence: 75%

Section: Function Of the Mcm2/5 Gate In The Mcm2-7 Double Hexamermentioning

confidence: 99%

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…Intrinsic to this is the functional loading of the MCM helicase complex, which is believed to involve ring opening between MCM subunits 2 and 5. 10, 11 Exactly how this is achieved on chromatin, and rendered functional by accurate interaction with both template and accessory factors, is not fully understood even in yeast.…”

Section: Introductionmentioning

confidence: 99%

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

et al. 2015

View full text Add to dashboard Cite

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.

show abstract

“…Biochemical evidence indicates that the gate-open Mcm2-7 conformation lacks helicase activity, while the gateclosed form retains activity (29). In vivo, the Mcm gate has two known functions: (i) to serve as an entry site for DNA loading during origin association (29) and (ii) to regulate DNA unwinding during G 1 /S.…”

mentioning

confidence: 99%

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate

Tsai

Vijayraghavan

Prinz

et al. 2015

Molecular and Cellular Biology

View full text Add to dashboard Cite

The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response. The eukaryotic DNA replication checkpoint (DRC) monitors chromosome duplication during S phase and, if irregularities occur, facilitates numerous cellular responses that promote genome stability. In budding yeast, this checkpoint depends upon a phosphorylation cascade initiated by the upstream sensor kinase Mec1/ATR (1, 2), which in turn leads to the phosphorylation and activation of the Mrc1/claspin mediator (3, 4) and ultimately the effector kinase Rad53/Chk1 (5, 6). During deoxynucleoside triphosphate (dNTP) limitation or other forms of replication stress, DRC activation protects genome stability by Rad53-dependent phosphorylation of multiple downstream targets that serve to stabilize nascent replication forks and blocks cell cycle progression, inappropriate recombination (7-9), and the activation of late origins until the stress is alleviated (reviewed in reference 10). In addition to the DRC, a second related pathway that specifically monitors and responds to DNA damage and double-strand breaks also operates during S phase (DNA damage checkpoint [DDC]) (reviewed in reference 11).How the DRC cascade mechanistically interacts with the core replication machinery is incompletely understood. Current evidence indicates that replication plays a passive role in the process. DNA lesions or stress causes a physical uncoupling between DNA polymerase and the replicative helicase; this in turn results in an aberrantly increased level of single-stranded DNA (ssDNA) production that leads to checkpoint activation (12-14). Correspondingly, normal replication fork formation is a prerequisite for DRC activation (15-18).However, strong interactions between DRC components and core replication factors, even in the absence of replication stress, suggest that DNA replication in general and the MCM replicative helicase in particular play broader roles in the DRC. The mediator proteins in the cascade (Mrc1/claspin, Tof1/Timeless, and Csm3/ Tipin) physically interact with and stabilize both Mcm2-7 and DNA polymerase ε (19-23) and protect fork integrity during replication stress (21,2...

show abstract

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2–7 onto DNA

Cited by 114 publications

References 55 publications

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate

Contact Info

Product

Resources

About