| 1. |
- Cisneros, David A., et al.
(författare)
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Heterologous assembly of type IV pili by a type II secretion system reveals the role of minor pilins in assembly initiation
- 2012
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 86:4, s. 805-818
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Tidskriftsartikel (refereegranskat)abstract
- In Gram-negative bacteria, type IV pilus assembly (T4PS) and type II secretion (T2SS) systems polymerize inner membrane proteins called major pilins or pseudopilins respectively, into thin filaments. Four minor pilins are required in both systems for efficient fibre assembly. Escherichia coli K-12 has a set of T4PS assembly genes that are silent under standard growth conditions. We studied the heterologous assembly of the E. coli type IV pilin PpdD by the Klebsiella oxytoca T2SS called the Pul system. PpdD pilus assembly in this context depended on the expression of the K. oxytoca minor pseudopilin genes pulHIJK or of the E. coli minor pilin genes ppdAB-ygdB-ppdC. The E. coli minor pilins restored assembly of the major pseudopilin PulG in a pulHIJK mutant, but not the secretion of the T2SS substrate pullulanase. Thus, minor pilins and minor pseudopilins are functionally interchangeable in initiating major pilin assembly, further extending the fundamental similarities between the two systems. The data suggest that, in both systems, minor pilins activate the assembly machinery through a common self-assembly mechanism. When produced together, PulG and PpdD assembled into distinct homopolymers, establishing major pilins as key determinants of pilus elongation and structure.
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| 2. |
- Madru, ClEment, et al.
(författare)
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Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA
- 2020
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 angstrom. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Pol alpha replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.
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| 3. |
- Mittler, Eva, et al.
(författare)
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Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses
- 2022
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Ingår i: Science Translational Medicine. - : American Association for the Advancement of Science. - 1946-6234 .- 1946-6242. ; 14:636
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Tidskriftsartikel (refereegranskat)abstract
- The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.
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| 4. |
- Raia, Pierre, et al.
(författare)
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Structure of the DP1-DP2 PolD complex bound with DNA and its implications for the evolutionary history of DNA and RNA polymerases
- 2019
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo-electron microscopy (cryo-EM) structure of the heterodimeric DP1-DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1-DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.
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