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Reproduction (Cambridge, England) Feb 2010Haploid male germ cells package their DNA into a volume that is typically 10% or less that of a somatic cell nucleus. To achieve this remarkable level of compaction,... (Review)
Review
Haploid male germ cells package their DNA into a volume that is typically 10% or less that of a somatic cell nucleus. To achieve this remarkable level of compaction, spermatozoa replace most of their histones with smaller, highly basic arginine and (in eutherians) cysteine rich protamines. One reason for such a high level of compaction is that it may help optimise nuclear shape and hence support the gametes' swimming ability for the long journey across the female reproductive tract to the oocyte. Super-compaction of the genome may confer additional protection from the effects of genotoxic factors. However, many species including the human retain a fraction of their chromatin in the more relaxed nucleosomal configuration that appears to run counter to the ergonomic, toroidal and repackaging of sperm DNA. Recent research suggests that the composition of this 'residual' nucleosomal compartment, a generally overlooked feature of the male gamete, is far more significant and important than previously thought. In this respect, the transport and incorporation of modified paternal histones by the spermatozoon to the zygote has been demonstrated and indicates another potential paternal effect in the epigenetic reprogramming of the zygote following fertilisation that is independent of imprinting status. In this review, the most recent research into mammalian spermatozoal chromatin composition is discussed alongside evidence for conserved, non-randomly located nucleosomal domains in spermatozoal nuclei, all supporting the hypothesis that the spermatozoon delivers a novel epigenetic signature to the egg that may be crucial for normal development. We also provide some thoughts on why this signature may be required in early embryogenesis.
Topics: Animals; Chromatin Assembly and Disassembly; DNA; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Developmental; Genetic Code; Genomic Imprinting; Histones; Humans; Male; Nucleosomes; Paternity; Protamines; Sperm-Ovum Interactions; Spermatogenesis; Spermatozoa
PubMed: 19759174
DOI: 10.1530/REP-09-0281 -
Annals of Biomedical Engineering Oct 2009Nanobiotechnology involves the creation, characterization, and modification of organized nanomaterials to serve as building blocks for constructing nanoscale devices in... (Review)
Review
Nanobiotechnology involves the creation, characterization, and modification of organized nanomaterials to serve as building blocks for constructing nanoscale devices in technology and medicine. Living systems contain a wide variety of nanomachines and highly ordered structures of macromolecules. The novelty and ingenious design of the bacterial virus phi29 DNA packaging motor and its parts inspired the synthesis of this motor and its components as biomimetics. This 30-nm nanomotor uses six copies of an ATP-binding pRNA to gear the motor. The structural versatility of pRNA has been utilized to construct dimers, trimers, hexamers, and patterned superstructures via the interaction of two interlocking loops. The approach, based on bottom-up assembly, has also been applied to nanomachine fabrication, pathogen detection and the delivery of drugs, siRNA, ribozymes, and genes to specific cells in vitro and in vivo. Another essential component of the motor is the connector, which contains 12 copies of a protein gp10 to form a 3.6-nm central channel as a path for DNA. This article will review current studies of the structure and function of the phi29 DNA packaging motor, as well as the mechanism of motion, the principle of in vitro construction, and its potential nanotechnological and medical applications.
Topics: Adenosine Triphosphate; Bacillus Phages; Capsid; DNA; DNA Packaging; Genetic Therapy; Molecular Motor Proteins; Nanomedicine; RNA
PubMed: 19495981
DOI: 10.1007/s10439-009-9723-0 -
Methods in Molecular Biology (Clifton,... 2005A controllable, 30-nm imitating DNA-packaging motor was constructed. The motor is driven by six synthetic adenosine triphosphate (ATP)-binding RNA (packaging RNA [pRNA])... (Review)
Review
A controllable, 30-nm imitating DNA-packaging motor was constructed. The motor is driven by six synthetic adenosine triphosphate (ATP)-binding RNA (packaging RNA [pRNA]) monomers, similar to the driving of a bolt with a hex nut. Conformational change and sequential action of the RNA with fivefold (viral capsid)/sixfold (pRNA hexamer) mismatch could ensure continuous rotation of the motor with ATP as energy. In the presence of ATP and magnesium, a 5-microm synthetic DNA was packaged using this motor. On average, one ATP was used to translocate two bases of DNA. The DNA-filled capsids were subsequently converted into up to 109 PFU/mL of infectious virus. The three-dimensional structures of pRNA monomer, dimer, and hexamer have been probed by photoaffinity crosslinking, chemical modification interference, cryo-atomic force microscopy, and computer modeling. The pRNA's size and shape can be controlled and manipulated at will to form stable dimers and trimers. Cryo-atomic force microscopy revealed that monomers, dimers, and trimers displayed a checkmark outline, elongated shape, and triangular structure, respectively. The motor can be turned off by gamma-S-ATP or EDTA and turned on again with the addition of ATP or magnesium, respectively. The formation of ordered structural arrays of the motor complex and its components, the retention of motor function after the 3'-end extension of the pRNA, and the ease of RNA dimer, trimer, and hexamer manipulation with desired shape and size make this RNA-containing motor a promising tool for drug and gene delivery and for use in nanodevices.
Topics: Adenosine Triphosphate; Bacillus Phages; Capsid; DNA; DNA Packaging; Genetic Therapy; Molecular Motor Proteins; Nanotechnology; RNA
PubMed: 15657489
DOI: 10.1385/1-59259-858-7:285 -
Current Opinion in Virology Apr 2014Structural information can inform our understanding of virus origins and evolution. The herpesviruses and tailed bacteriophages constitute two large families of dsDNA... (Comparative Study)
Comparative Study Review
Structural information can inform our understanding of virus origins and evolution. The herpesviruses and tailed bacteriophages constitute two large families of dsDNA viruses which infect vertebrates and prokaryotes respectively. A relationship between these disparate groups was initially suggested by similarities in their capsid assembly and DNA packaging strategies. This relationship has now been confirmed by a range of studies that have revealed common structural features in their capsid proteins, and similar organizations and sequence conservation in their DNA packaging machinery and maturational proteases. This concentration of conserved traits in proteins involved in essential and primordial capsid/packaging functions is evidence that these structures are derived from an ancient, common ancestor and is in sharp contrast to the lack of such evidence for other virus functions.
Topics: Animals; Bacteriophages; DNA Packaging; DNA Viruses; Herpesviridae; Herpesviridae Infections; Humans; Virus Assembly
PubMed: 24747680
DOI: 10.1016/j.coviro.2014.02.003 -
Viruses May 2020A "DNA crunching" linear motor mechanism that employs a grip-and-release transient spring like compression of B- to A-form DNA has been found in our previous studies....
A "DNA crunching" linear motor mechanism that employs a grip-and-release transient spring like compression of B- to A-form DNA has been found in our previous studies. Our FRET measurements in vitro show a decrease in distance from TerL to portal during packaging; furthermore, there is a decrease in distance between closely positioned dye pairs in the Y-stem of translocating Y-DNA that conforms to B- and A- structure. In normal translocation into the prohead the TerL motor expels all B-form tightly binding YOYO-1 dye that cannot bind A-form. The TerL motor cannot package A-form dsRNA. Our work reported here shows that addition of helper B form DNA:DNA (D:D) 20mers allows increased packaging of heteroduplex A-form DNA:RNA 20mers (D:R), evidence for a B- to A-form spring motor pushing duplex nucleic acid. A-form DNA:RNA 25mers, 30mers, and 35mers alone are efficiently packaged into proheads by the TerL motor showing that a proposed hypothetical dehydration motor mechanism operating on duplex substrates does not provide the packaging motor force. Taken together with our previous studies showing TerL motor protein motion toward the portal during DNA packaging, our present studies of short D:D and D:R duplex nucleic acid substrates strongly supports our previous evidence that the protein motor pushes rather than pulls or dehydrates duplex substrates to provide the translocation into prohead packaging force.
Topics: Bacteriophage T4; DNA Packaging; DNA, Viral; Dehydration; Endodeoxyribonucleases; Nucleic Acid Conformation; Viral Proteins
PubMed: 32397493
DOI: 10.3390/v12050522 -
Journal of Molecular Biology Sep 2008Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA...
Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.
Topics: Adenosine Triphosphatases; Bacillus Phages; Binding Sites; Capsid; Cryoelectron Microscopy; DNA; DNA Packaging; DNA-Binding Proteins; Molecular Motor Proteins; Nucleic Acid Conformation; Protein Structure, Quaternary; RNA, Viral; Ribonucleases; Solubility
PubMed: 18674782
DOI: 10.1016/j.jmb.2008.04.034 -
The Enzymes 2021Although the process of genome encapsidation is highly conserved in tailed bacteriophages and eukaryotic double-stranded DNA viruses, there are two distinct packaging... (Review)
Review
Although the process of genome encapsidation is highly conserved in tailed bacteriophages and eukaryotic double-stranded DNA viruses, there are two distinct packaging pathways that these viruses use to catalyze ATP-driven translocation of the viral genome into a preassembled procapsid shell. One pathway is used by ϕ29-like phages and adenoviruses, which replicate and subsequently package a monomeric, unit-length genome covalently attached to a virus/phage-encoded protein at each 5'-end of the dsDNA genome. In a second, more ubiquitous packaging pathway characterized by phage lambda and the herpesviruses, the viral DNA is replicated as multigenome concatemers linked in a head-to-tail fashion. Genome packaging in these viruses thus requires excision of individual genomes from the concatemer that are then translocated into a preassembled procapsid. Hence, the ATPases that power packaging in these viruses also possess nuclease activities that cut the genome from the concatemer at the beginning and end of packaging. This review focuses on proposed mechanisms of genome packaging in the dsDNA viruses using unit-length ϕ29 and concatemeric λ genome packaging motors as representative model systems.
Topics: Bacteriophage lambda; DNA Packaging; DNA, Viral; Viral Genome Packaging; Virus Assembly
PubMed: 34861943
DOI: 10.1016/bs.enz.2021.09.006 -
Protein & Cell May 2020
Topics: Bacteriophages; DNA Packaging; DNA, Viral; Models, Molecular
PubMed: 32266588
DOI: 10.1007/s13238-020-00715-9 -
Advances in Experimental Medicine and... 2012Many double-stranded DNA bacteriophages and viruses use specialized ATP-driven molecular machines to package their genomes into tightly confined procapsid shells. Over... (Review)
Review
Many double-stranded DNA bacteriophages and viruses use specialized ATP-driven molecular machines to package their genomes into tightly confined procapsid shells. Over the last decade, single-molecule approaches - and in particular, optical tweezers - have made key contributions to our understanding of this remarkable process. In this chapter, we review these advances and the insights they have provided on the packaging mechanisms of three bacteriophages: φ 29, λ, and T4.
Topics: Adenosine Triphosphate; Bacteriophages; DNA Packaging; DNA, Viral; Models, Molecular; Molecular Motor Proteins; Optical Tweezers; Protein Conformation; Protein Subunits; Viral Proteins; Virus Assembly
PubMed: 22297530
DOI: 10.1007/978-1-4614-0980-9_24 -
Physical Review Letters Jun 2014We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions...
We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine(3+) causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation.
Topics: Bacteriophages; DNA Packaging; DNA, Viral; Nucleic Acid Conformation; Optical Tweezers
PubMed: 24996111
DOI: 10.1103/PhysRevLett.112.248101