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Nature Communications Nov 2021Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors...
Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.
Topics: Adenosine Triphosphatases; Bacteriophage T4; DNA Packaging; DNA, Viral; Viral Genome Packaging; Viral Proteins; Virus Assembly
PubMed: 34772936
DOI: 10.1038/s41467-021-26800-z -
PLoS Pathogens Dec 2019A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes...
A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes involved in this process are the terminases. The HCMV terminase complex consists of two terminase subunits, the ATPase pUL56 and the nuclease pUL89. A potential third component pUL51 has been proposed. Even though the terminase subunit pUL89 has been shown to be essential for DNA packaging and interaction with pUL56, it is not known how pUL89 mechanistically achieves sequence-specific DNA binding and nicking. To identify essential domains and invariant amino acids vis-a-vis nuclease activity and DNA binding, alanine substitutions of predicted motifs were analyzed. The analyses indicated that aspartate 463 is an invariant amino acid for the nuclease activity, while argine 544 is an invariant aa for DNA binding. Structural analysis of recombinant protein using electron microscopy in conjunction with single particle analysis revealed a curvilinear monomer with two distinct domains connected by a thinner hinge-like region that agrees well with the predicted structure. These results allow us to model how the terminase subunit pUL89's structure may mediate its function.
Topics: Cytomegalovirus; DNA Packaging; Protein Conformation; Structure-Activity Relationship; Viral Proteins
PubMed: 31809525
DOI: 10.1371/journal.ppat.1008175 -
Proceedings of the National Academy of... May 2022A molecular architecture is proposed for a representative mitotic chromosome, human chromosome 10. This architecture is built on an interphase chromosome structure based...
A molecular architecture is proposed for a representative mitotic chromosome, human chromosome 10. This architecture is built on an interphase chromosome structure based on cryo-electron microscopy (cryo-EM) cellular tomography [J. Sedat et al., Proc. Natl. Acad. Sci. U.S.A., in press], thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle for mitotic chromosomes is specific coiling of the 11-nm nucleosome fiber into large scale, ∼200-nm interphase structures, a Slinky [https://en.wikipedia.org/wiki/Slinky; motif cited in S. Bowerman et al., eLife 10, e65587 (2021)], then further modified with subsequent additional coiling for the final mitotic chromosome structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well-known cytological configurations. In addition, proof is experimentally provided by digital PCR technology that G1 T cell nuclei are diploid with one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.
Topics: Cell Nucleus; Chromosomes, Human, Pair 10; DNA Packaging; G1 Phase; Humans; Mitosis; Nucleosomes; Polymerase Chain Reaction; T-Lymphocytes
PubMed: 35544689
DOI: 10.1073/pnas.2119107119 -
International Journal of Molecular... Jul 2017Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome... (Review)
Review
Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage.
Topics: Animals; Chromatin Assembly and Disassembly; DNA Damage; DNA Repair; DNA Replication; Genomic Instability; Humans
PubMed: 28698521
DOI: 10.3390/ijms18071486 -
Neurotherapeutics : the Journal of the... Oct 2013The integrity of the genome is continuously challenged by both endogenous and exogenous DNA damaging agents. Neurons, due to their post-mitotic state, high metabolism,... (Review)
Review
The integrity of the genome is continuously challenged by both endogenous and exogenous DNA damaging agents. Neurons, due to their post-mitotic state, high metabolism, and longevity are particularly prone to the accumulation of DNA lesions. Indeed, DNA damage has been suggested as a major contributor to both age-associated neurodegenerative diseases and acute neurological injury. The DNA damage response is a key factor in maintaining genome integrity. It relies on highly dynamic posttranslational modifications of the chromatin and DNA repair proteins to allow signaling, access, and repair of the lesion. Drugs that modulate the activity of the enzymes responsible for these modifications have emerged as attractive therapeutic compounds to treat neurodegeneration. In this review, we discuss the role of DNA double-strand breaks and abnormal chromatin modification patterns in a range of neurodegenerative conditions, and the chromatin modifiers that might ameliorate them. Finally, we suggest that understanding the epigenetic modifications specific to neuronal DNA repair is crucial for the development of efficient neurotherapeutic strategies.
Topics: Chromatin Assembly and Disassembly; DNA Breaks, Double-Stranded; DNA Repair; Humans; Neurodegenerative Diseases
PubMed: 24072514
DOI: 10.1007/s13311-013-0210-9 -
Journal of the Royal Society, Interface Jun 2021One of the least understood properties of chromatin is the ability of its similar regions to recognize each other through weak interactions. Theories based on...
One of the least understood properties of chromatin is the ability of its similar regions to recognize each other through weak interactions. Theories based on electrostatic interactions between helical macromolecules suggest that the ability to recognize sequence homology is an innate property of the non-ideal helical structure of DNA. However, this theory does not account for the nucleosomal packing of DNA. Can homologous DNA sequences recognize each other while wrapped up in the nucleosomes? Can structural homology arise at the level of nucleosome arrays? Here, we present a theoretical model for the recognition potential well between chromatin fibres sliding against each other. This well is different from the one predicted for bare DNA; the minima in energy do not correspond to literal juxtaposition, but are shifted by approximately half the nucleosome repeat length. The presence of this potential well suggests that nucleosome positioning may induce mutual sequence recognition between chromatin fibres and facilitate the formation of chromatin nanodomains. This has implications for nucleosome arrays enclosed between CTCF-cohesin boundaries, which may form stiffer stem-like structures instead of flexible entropically favourable loops. We also consider switches between chromatin states, e.g. through acetylation/deacetylation of histones, and discuss nucleosome-induced recognition as a precursory stage of genetic recombination.
Topics: Chromatin; Chromatin Assembly and Disassembly; DNA; Histones; Nucleosomes
PubMed: 34129789
DOI: 10.1098/rsif.2021.0147 -
Nature Reviews. Genetics Apr 2015The variation in local rates of mutations can affect both the evolution of genes and their function in normal and cancer cells. Deciphering the molecular determinants of... (Review)
Review
The variation in local rates of mutations can affect both the evolution of genes and their function in normal and cancer cells. Deciphering the molecular determinants of this variation will be aided by the elucidation of distinct types of mutations, as they differ in regional preferences and in associations with genomic features. Chromatin organization contributes to regional variation in mutation rates, but its contribution differs among mutation types. In both germline and somatic mutations, base substitutions are more abundant in regions of closed chromatin, perhaps reflecting error accumulation late in replication. By contrast, a distinctive mutational state with very high levels of insertions and deletions (indels) and substitutions is enriched in regions of open chromatin. These associations indicate an intricate interplay between the nucleotide sequence of DNA and its dynamic packaging into chromatin, and have important implications for current biomedical research. This Review focuses on recent studies showing associations between chromatin state and mutation rates, including pairwise and multivariate investigations of germline and somatic (particularly cancer) mutations.
Topics: Animals; Chromatin Assembly and Disassembly; Evolution, Molecular; Genetic Variation; Genome; Humans; Mutation Rate
PubMed: 25732611
DOI: 10.1038/nrg3890 -
IUBMB Life Nov 2016Cells all share essentially the same genome, which is shaped by the developmental program to bring about various outcomes. It is well known that epigenetic regulation is... (Review)
Review
Cells all share essentially the same genome, which is shaped by the developmental program to bring about various outcomes. It is well known that epigenetic regulation is a key process in determining the different cell functional output from the genetic information. Somatic reprogramming is a dramatic demonstration of this effect, and has opened the gate to the investigation of cell fate determination as cells reverse the developmental program. These studies have revealed which epigenetic marks set during normal development are important for cell specification. Here, we review the epigenetic landmarks that cells pass through during somatic reprogramming, and give an overview to the many remaining unclear epigenetic regulatory events that occurs during reprogramming. © 2016 IUBMB Life, 68(11):854-857, 2016.
Topics: Acetylation; Animals; Cell Differentiation; Chromatin Assembly and Disassembly; DNA Methylation; Epigenesis, Genetic; Humans; Protein Processing, Post-Translational
PubMed: 27785904
DOI: 10.1002/iub.1577 -
Nucleic Acids Research Jan 2013The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal...
The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal ATPase activity that fuels DNA translocation during packaging and a C-terminal nuclease activity required for initiation and termination of the packaging cycle. Here, we report that bacteriophage SPP1 large terminase (gp2) is a metal-dependent nuclease whose stability and activity are strongly and preferentially enhanced by Mn(2+) ions. Mutation of conserved residues that coordinate Mn(2+) ions in the nuclease catalytic site affect the metal-induced gp2 stabilization and impair both gp2-specific cleavage at the packaging initiation site pac and unspecific nuclease activity. Several of these mutations block also DNA encapsidation without affecting ATP hydrolysis or gp2 C-terminus binding to the procapsid portal vertex. The data are consistent with a mechanism in which the nuclease domain bound to the portal switches between nuclease activity and a coordinated action with the ATPase domain for DNA translocation. This switch of activities of the nuclease domain is critical to achieve the viral chromosome packaging cycle.
Topics: Adenosine Triphosphatases; Bacillus Phages; Capsid; Catalytic Domain; Cations, Divalent; DNA Cleavage; DNA Packaging; Endodeoxyribonucleases; Manganese; Metals; Mutation; Phenotype; Protein Structure, Tertiary; Substrate Specificity; Viral Proteins
PubMed: 23118480
DOI: 10.1093/nar/gks974 -
Biophysical Journal Jan 2016During replication of the ϕ29 bacteriophage inside a bacterial host cell, a DNA packaging motor transports the viral DNA into the procapsid against a pressure...
During replication of the ϕ29 bacteriophage inside a bacterial host cell, a DNA packaging motor transports the viral DNA into the procapsid against a pressure difference of up to 40 ± 20 atm. Several models have been proposed for the underlying molecular mechanism. Here we have used molecular dynamics simulations to examine the role of the connector part of the motor, and specifically the one-way revolution and the push-roll model. We have focused at the structure and intermolecular interactions between the DNA and the connector, for which a near-complete structure is available. The connector is found to induce considerable DNA deformations with respect to its canonical B-form. We further assessed by force-probe simulations to which extent the connector is able to prevent DNA leakage and found that the connector can act as a partial one-way valve by a check-valve mechanism via its mobile loops. Analysis of the geometry, flexibility, and energetics of channel lysine residues suggested that this arrangement of residues is incompatible with the observed DNA packaging step-size of ∼2.5 bp, such that the step-size is probably determined by the other components of the motor. Previously proposed DNA revolution and rolling motions inside the connector channel are both found implausible due to structural entanglement between the DNA and connector loops that have not been resolved in the crystal structure. Rather, in the simulations, the connector facilitates minor DNA rotation during the packaging process compatible with recent optical-tweezers experiments. Combined with the available experimental data, our simulation results suggest that the connector acts as a check-valve that prevents DNA leakage and induces DNA compression and rotation during DNA packaging.
Topics: Amino Acid Sequence; Base Sequence; Capsid Proteins; DNA Packaging; DNA, Viral; Molecular Dynamics Simulation; Molecular Sequence Data; Protein Binding; Protein Structure, Tertiary; Rotation
PubMed: 26789768
DOI: 10.1016/j.bpj.2015.12.010