A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template

Boudet, Julien; Devillier, Jean-Christophe; Wiegand, Thomas; Salmon, Loïc; Meier, Beat H.; Lipps, Georg; Allain, Frédéric H.‐T.

doi:10.1016/j.cell.2018.11.031

Cited by 24 publications

(71 citation statements)

References 51 publications

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“…To be sure, replication initiation involves a multiprotein complex, with, beside DnaA, helicase PriA as another ATPdependent factor that could act as a further discriminating MxD or possibly as a mechanical transducer (Windgassen et al, 2018). DNA primase, that starts replication, might even combine recognition of the target initiation site with formation of a first dinucleotide primer made from the two ATP molecules used in the process, an economical way to make use of ATP in the process (Boudet et al, 2019).…”

Section: Other Processes Working Out Specific Features Of Nucleic Acidsmentioning

confidence: 99%

Omnipresent Maxwell's demons orchestrate information management in living cells

Boël

Danot

Lorenzo

et al. 2019

Microbial Biotechnology

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Summary The development of synthetic biology calls for accurate understanding of the critical functions that allow construction and operation of a living cell. Besides coding for ubiquitous structures, minimal genomes encode a wealth of functions that dissipate energy in an unanticipated way. Analysis of these functions shows that they are meant to manage information under conditions when discrimination of substrates in a noisy background is preferred over a simple recognition process. We show here that many of these functions, including transporters and the ribosome construction machinery, behave as would behave a material implementation of the information‐managing agent theorized by Maxwell almost 150 years ago and commonly known as Maxwell's demon (MxD). A core gene set encoding these functions belongs to the minimal genome required to allow the construction of an autonomous cell. These MxDs allow the cell to perform computations in an energy‐efficient way that is vastly better than our contemporary computers.

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Section: Other Processes Working Out Specific Features Of Nucleic Acidsmentioning

confidence: 99%

Omnipresent Maxwell's demons orchestrate information management in living cells

Boël

Danot

Lorenzo

et al. 2019

Microbial Biotechnology

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“…Sample preparation by sedimentation allowed for the investigation of proteins that are difficult (or even impossible) to crystallize. Theoretically, the molecular mass of the protein determines the success of sedimentation, and proteins, such as the RNA polymerase subunits Rpo4/7 * (the * indicates that the Rpo7 unit is uniformly 13 C/ 15 N labeled) with a molecular weight of 34 kDa (Torosyan et al, 2019), the pRN1 primase with 40 kDa (Boudet et al, 2019), the neonatal Fc receptor with 40 kDa (Stöppler et al, 2018) as well as the human superoxide dismutase with 32 kDa (Fragai et al, 2013), the bacterial helicase DnaB with 708 kDa (Gardiennet et al, 2012), the iron-storage protein ferritin with 480 kDa (Bertini et al, 2012), a variety of supramolecular assemblies (Lecoq et al, 2018;Gauto et al, 2019), and PEGylated proteins (Ravera et al, 2016), were shown to form sediments suitable for solid-state NMR. This is for some of these systems probably a consequence of oligomerization that has been induced by the high protein concentrations (Bertini et al, 2013;Fragai et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…Here, we describe our experimental observations regarding the stability of four sedimented samples from different protein systems: (i) the bacterial helicase DnaB from Helicobacter pylori in a complex with ADP:AlF 4 − and DNA (Wiegand et al, 2019), (ii) the archaeal pRN1 primase from Sulfolobus islandicus in a complex with DNA and ATP (Boudet et al, 2019), (iii) the membrane protein BmrA, an ABC transporter from Bacillus subtilis (Lacabanne et al, 2019b), and (iv) the two RNA polymerase subunits Rpo4/7 * from Methanocaldococcus jannaschii. We use 2D DARR spectra and 31 P cross polarization (CP) MAS experiments to assess the long-term stability of these sedimented protein samples.…”

Section: Introductionmentioning

confidence: 99%

Sedimentation Yields Long-Term Stable Protein Samples as Shown by Solid-State NMR

Wiegand

Lacabanne

Torosyan

et al. 2020

Front. Mol. Biosci.

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Today, the sedimentation of proteins into a magic-angle spinning (MAS) rotor gives access to fast and reliable sample preparation for solid-state Nuclear Magnetic Resonance (NMR), and this has allowed for the investigation of a variety of noncrystalline protein samples. High protein concentrations on the order of 400 mg/mL can be achieved, meaning that around 50-60% of the NMR rotor content is protein; the rest is a buffer solution, which includes counter ions to compensate for the charge of the protein. We have demonstrated herein the long-term stability of four sedimented proteins and complexes thereof with nucleotides, comprising a bacterial DnaB helicase, an ABC transporter, an archaeal primase, and an RNA polymerase subunit. Solid-state NMR spectra recorded directly after sample filling and up to 5 years later indicated no spectral differences and no loss in signal intensity, allowing us to conclude that protein sediments in the rotor can be stable over many years. We have illustrated, using an example of an ABC transporter, that not only the structure is maintained, but that the protein is still functional after long-term storage in the sedimented state.

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“…A nuclear magnetic resonance study on the primase part of primase-polymerase ORF904 from archaeal plasmid pRN1 showed the helix bundle domain of ORF904 bound to template DNA and two ATP molecules, suggesting that this structure represents an early step of the catalytic cycle before the template DNA and the two nucleotides are transferred to the catalytic domain for dinucleotide formation (17).…”

Section: Introductionmentioning

confidence: 99%

The helix bundle domain of primase RepB’ is required for dinucleotide formation and extension

Banchenko

Weise

Lanka

et al. 2020

Preprint

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During DNA replication, primases synthesize oligonucleotide primers on single stranded template DNA, which are then extended by DNA polymerases to synthesize a complementary DNA strand. Primase RepB' of plasmid RSF1010 initiates DNA replication on two 40 nucleotide long inverted repeats, termed ssiA and ssiB, within the oriV of RSF1010. RepB' consists of a catalytic domain and a helix bundle domain which are connected by long α-helix 6 and an unstructured linker. Previous work has demonstrated that RepB' requires both domains for initiation of dsDNA synthesis in DNA replication assays. However, the precise functions of these two domains in primer synthesis have been unknown. Here we report, that both domains of RepB' are required to synthesizes a 10-12 nucleotide long DNA primer whereas the isolated domains are inactive. Mutational analysis of the catalytic domain indicates that the solvent exposed W50 plays a critical role in resolving a hairpin structure formed by ssiA/ssiB. Three structurally conserved aspartates (D77, D78 and D134) of RepB' catalyse nucleotidyltransferase activity. Mutations on the helix bundle domain are identified that either reduce the primer length to a dinucleotide (R285A) or abolish primer synthesis (D238A) indicating that the helix bundle domain is required to form and extend the initial dinucleotide synthesized by the catalytic domain.

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A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template

Cited by 24 publications

References 51 publications

Omnipresent Maxwell's demons orchestrate information management in living cells

Omnipresent Maxwell's demons orchestrate information management in living cells

Sedimentation Yields Long-Term Stable Protein Samples as Shown by Solid-State NMR

The helix bundle domain of primase RepB’ is required for dinucleotide formation and extension

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