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Special Issue "Protein Machines Acting on DNA: Structure, Dynamics and Functional Mechanisms"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (31 March 2021).

Special Issue Editor

Dr. Carlos Penedo
E-Mail Website
Guest Editor
University of St Andrews, United Kingdom Laboratory for Biophysics and Biomolecular Dynamics Centre of Biophotonics, School of Physics and Astronomy, KY16 9SS Biomolecular Sciences Research Complex (BSRC), School of Biology. KY16 9ST

Special Issue Information

Dear Colleagues,

The interaction between individual proteins and multi-protein complexes with DNA sequences is crucial for cell survival across all domains of life. These interactions are involved in a range of processes including DNA condensation, transcription, replication and repair and have received  considerable attention due to their biological relevance. Over the last decade, our understanding of how these protein machineries physically interact with the nucleic acid structure to preserve DNA integrity, regulate its expression and resolve damaged sequences has progressed enormously. These advances have taken place at all mechanistic steps in which such interactions can be dissected: nucleic acid recognition, assembly, conformational reorganization, recruitment of additional partners and chemical catalysis. Such progress has been facilitated by parallel breakthroughs in many areas including DNA sequencing technologies to map interactions across the genome, cryo-EM methods to unveil structures of difficult-to-crystallize macro-complexes, more versatile site-specific tagging and cross-linking strategies, and the ability to detect low-populated transient states with sub-millisecond resolution at single-molecule level. As a result, molecular-level information regarding protein diffussion along the genome, dynamic assembly of these complexes and enzymatic aspects has never been so detailed both in vitro and in vivo. Moreover, the ability to edit specific DNA sequences, with unprecedented simplicity, using CRISPR-based technologies opens a new era to investigate genome-based functionality and anomalies. Articles in this special issue should focus on structural aspects and/or kinetic insights regarding the functional mechanisms of protein-DNA interactions. Papers providing molecular-level information or techniques enabling this would be of special interest.

Dr. Carlos Penedo
Guest Editor

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

  • Nucleic acids recognition and processing
  • Structure determination by Cryo-EM/X-ray
  • Dynamics of protein-DNA complexes in vivo and in vitro
  • DNA replication
  • DNA transcription
  • DNA repair
  • DNA recombination

Published Papers (4 papers)

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Research

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Article
A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity
Int. J. Mol. Sci. 2021, 22(6), 2848; https://doi.org/10.3390/ijms22062848 - 11 Mar 2021
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Abstract
Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational [...] Read more.
Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells. Full article
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Article
Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription
Int. J. Mol. Sci. 2021, 22(5), 2398; https://doi.org/10.3390/ijms22052398 - 27 Feb 2021
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Abstract
Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters [...] Read more.
Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion. Full article
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Review

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Review
Molecular Mechanisms Regulating the DNA Repair Protein APE1: A Focus on Its Flexible N-Terminal Tail Domain
Int. J. Mol. Sci. 2021, 22(12), 6308; https://doi.org/10.3390/ijms22126308 - 11 Jun 2021
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Abstract
APE1 (DNA (apurinic/apyrimidinic site) endonuclease 1) is a key enzyme of one of the major DNA repair routes, the BER (base excision repair) pathway. APE1 fulfils additional functions, acting as a redox regulator of transcription factors and taking part in RNA metabolism. The [...] Read more.
APE1 (DNA (apurinic/apyrimidinic site) endonuclease 1) is a key enzyme of one of the major DNA repair routes, the BER (base excision repair) pathway. APE1 fulfils additional functions, acting as a redox regulator of transcription factors and taking part in RNA metabolism. The mechanisms regulating APE1 are still being deciphered. Structurally, human APE1 consists of a well-characterized globular catalytic domain responsible for its endonuclease activity, preceded by a conformationally flexible N-terminal extension, acquired along evolution. This N-terminal tail appears to play a prominent role in the modulation of APE1 and probably in BER coordination. Thus, it is primarily involved in mediating APE1 localization, post-translational modifications, and protein–protein interactions, with all three factors jointly contributing to regulate the enzyme. In this review, recent insights on the regulatory role of the N-terminal region in several aspects of APE1 function are covered. In particular, interaction of this region with nucleophosmin (NPM1) might modulate certain APE1 activities, representing a paradigmatic example of the interconnection between various regulatory factors. Full article
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Review
PARP Power: A Structural Perspective on PARP1, PARP2, and PARP3 in DNA Damage Repair and Nucleosome Remodelling
Int. J. Mol. Sci. 2021, 22(10), 5112; https://doi.org/10.3390/ijms22105112 - 12 May 2021
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Abstract
Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in [...] Read more.
Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in the DNA damage response and nucleosome remodelling. Activation of PARP happens through DNA binding via zinc fingers and/or the WGR domain. Modulation of their activity using PARP inhibitors occupying the NAD+ binding site has proven successful in cancer therapies. For decades, studies set out to elucidate their full-length molecular structure and activation mechanism. In the last five years, significant advances have progressed the structural and functional understanding of PARP1-3, such as understanding allosteric activation via inter-domain contacts, how PARP senses damaged DNA in the crowded nucleus, and the complementary role of histone PARylation factor 1 in modulating the active site of PARP. Here, we review these advances together with the versatility of PARP domains involved in DNA binding, the targets and shape of PARylation and the role of PARPs in nucleosome remodelling. Full article
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