Helicase loading: How to build a MCM2-7 double-hexamer

Riera, Alberto; Tognetti, Silvia; Speck, Christian

doi:10.1016/j.semcdb.2014.03.008

Cited by 23 publications

(16 citation statements)

References 65 publications

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“…Initiation of DNA replication is a multistep reaction that is carefully choreographed to promote replication fork assembly and regulated firing of replication origins (Costa et al 2013;Riera et al 2014;Tognetti et al 2015;Bell and Labib 2016;O'Donnell and Li 2016;Pellegrini and Costa 2016;Riera and Speck 2016;Bleichert et al 2017). Moreover, this process is highly regulated in order to coordinate DNA synthesis with the cell cycle and the energy status of the cell.…”

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confidence: 99%

From structure to mechanism—understanding initiation of DNA replication

et al. 2017

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DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.

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From structure to mechanism—understanding initiation of DNA replication

et al. 2017

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“…The mechanism for loading of hexameric helicase onto and encircling of DNA is still unknown, but it most likely requires origin-binding proteins and helicase accessory proteins to distort the origin of replication and pry open the hexameric helicase (51)(52)(53). Maintaining contact with the excluded strand while encircling the translocating strand would be more effective in sustaining an open dsDNA bubble for loading helicases.…”

Section: Discussionmentioning

confidence: 99%

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase

Graham

Tao

Dodge

et al. 2016

Journal of Biological Chemistry

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The archaeal minichromosomal maintenance (MCM) helicase from Sulfolobus solfataricus (SsoMCM) is a model for understanding structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. We have previously proposed an extension of the traditional steric exclusion model of unwinding to also include significant contributions with the excluded strand during unwinding, termed steric exclusion and wrapping (SEW). The SEW model hypothesizes that the displaced single strand tracks along paths on the exterior surface of hexameric helicases to protect singlestranded DNA (ssDNA) and stabilize the complex in a forward unwinding mode. Using hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance MS, we have probed the binding sites for ssDNA, using multiple substrates targeting both the encircled and excluded strand interactions. In each experiment, we have obtained >98.7% sequence coverage of SsoMCM from >650 peptides (5-30 residues in length) and are able to identify interacting residues on both the interior and exterior of SsoMCM. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identify binding sites for ssDNA during SsoMCM unwinding as well as validating the importance of the SEW model for hexameric helicase unwinding.DNA unwinding by hexameric replicative helicases is required for DNA replication elongation, providing the singlestranded DNA (ssDNA) 5 templates for leading and lagging strand synthesis. These hexameric helicase complexes form ring-like structures that preferentially encircle one of the DNA strands, providing stability in DNA binding and enhancing processivity for unwinding long stretches of DNA. In the steric exclusion model for unwinding, the motor domains within the central channel translocate along the encircled ssDNA while physically excluding the complementary ssDNA on the exterior. Although DNA unwinding by helicases can be effectively measured in gel-based and fluorescence assays, the specific molecular interactions with DNA are poorly described. Previously, we have proposed that the excluded strand is not a passive component of unwinding; rather, it will interact along specific exterior paths on the hexameric helicase surface to both stabilize the complex and promote forward unwinding (1). Although structures of hexameric helicases that include small stretches of ssDNA have been solved recently (2, 3), the interactions are constrained to central channel residues, and no molecular information is available at the duplex region or with respect to the excluded strand.Hexameric DNA replication helicases are known to exist across the three domains of life, including viruses. Althou...

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“…Eukaryotic cells have evolved a sophisticated machinery to achieve efficient helicase recruitment and loading. In S. cerevisiae eight protein factors make up the helicase loading machine: the sixsubunit origin recognition complex (ORC1-6), Cdc6 and Cdt1 (Riera et al 2014).…”

Section: Dna Licensingmentioning

confidence: 99%

“…This OCM complex is an intermediate of the pre-RC reaction and is, in contrast to the OCCM, able to recruit a second MCM2-7 hexamer to the DNA. Once the second MCM2-7 hexamer is recruited, a stable hexamer-hexamer interface is established (Evrin et al 2014), which leads to formation of a head-to-head MCM2-7 double-hexamer (Riera et al 2014). A summary of the known protein-protein interactions involved in S. cerevisiae pre-RC formation can be found in Table 1.…”

Section: Dna Licensingmentioning

confidence: 99%

Switch on the engine: how the eukaryotic replicative helicase MCM2–7 becomes activated

2014

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A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2-7 (MCM2-7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2-7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2-7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation;we focus on protein interactions during CMG formation, discuss structural changes during helicase activation and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.3

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Helicase loading: How to build a MCM2-7 double-hexamer

Cited by 23 publications

References 65 publications

From structure to mechanism—understanding initiation of DNA replication

From structure to mechanism—understanding initiation of DNA replication

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase

Switch on the engine: how the eukaryotic replicative helicase MCM2–7 becomes activated

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