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In human cells, generally a single mitochondrial DNA (mtDNA) is compacted into a nucleoprotein complex denoted the nucleoid. Each cell contains hundreds of nucleoids, which tend to cluster into small groups. It is unknown whether all nucleoids are equally involved in mtDNA replication and transcription or whether distinct nucleoid subpopulations exist. Here, we use multi-color STED super-resolution microscopy to determine the activity of individual nucleoids in primary human cells. We demonstrate that only a minority of all nucleoids are active. Active nucleoids are physically larger and tend to be involved in both replication and transcription. Inactivity correlates with a high ratio of the mitochondrial transcription factor A (TFAM) to the mtDNA of the individual nucleoid, suggesting that TFAM-induced nucleoid compaction regulates nucleoid replication and transcription activity in vivo. We propose that the stable population of highly compacted inactive nucleoids represents a storage pool of mtDNAs with a lower mutational load., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2021. The Author(s).)

The human adenovirus (HAdV) protein IX (pIX) is a minor component of the capsid that acts in part to stabilize the hexon-hexon interactions within the mature capsid. Virions lacking pIX have a reduced DNA packaging capacity and exhibit thermal instability. More recently, pIX has been developed as a platform for presentation of large polypeptides, such as fluorescent proteins or large targeting ligands, on the viral capsid. It is not known whether such modifications affect the natural ability of pIX to stabilize the HAdV virion. In this study, we show that addition of large polypeptides to pIX does not alter the natural stability of virions containing sub-wild-type-sized genomes. However, similar virions containing wild-type-sized genomes tend to genetically rearrange, likely due to selective pressure caused by virion instability as a result of compromised pIX function. IMPORTANCE Human adenovirus capsid protein IX (pIX) is involved in stabilizing the virion but has also been developed as a platform for presentation of various polypeptides on the surface of the virion. Whether such modifications affect the ability of pIX to stabilize the virion is unknown. We show that addition of large polypeptides to pIX can reduce both the DNA packaging capacity and the heat stability of the virion, which provides important guidance for the design of pIX-modified vectors., (Copyright © 2020 American Society for Microbiology.)

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., Competing Interests: The authors have declared that no competing interests exist.

Polycomb group proteins (PcGs) control the epigenetic and transcriptional state of developmental genes and regulatory elements during mammalian embryogenesis. Moreover, PcGs can also contribute to 3D genome organization, adding an additional layer of complexity to their regulatory functions. Understanding the mechanistic basis and the dynamics of PcG-dependent chromatin structures will help us untangle the full complexity of PcG function during development. Since most studies concerning the 3D organization of PcG-bound chromatin in mammals have been performed in embryonic stem cells (ESCs), here we will focus on this cell type characterized by its unique self-renewal and pluripotency properties. More specifically, we will highlight recent findings and discuss open questions regarding how PcG-dependent changes in 3D chromatin architecture control gene expression, cellular identity and differentiation potential in ESCs. We believe that this can serve to illustrate the diverse regulatory mechanisms by which PcG proteins control the proper execution of gene expression programs during mammalian embryogenesis., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We present the genome sequences of Salmonella enterica tailed phages Sasha, Sergei, and Solent. These phages, along with Salmonella phages 9NA, FSL_SP-062, and FSL_SP-069 and the more distantly related Proteus phage PmiS-Isfahan, have similarly sized genomes of between 52 and 57 kbp in length that are largely syntenic. Their genomes also show substantial genome mosaicism relative to one another, which is common within tailed phage clusters. Their gene content ranges from 80 to 99 predicted genes, of which 40 are common to all seven and form the core genome, which includes all identifiable virion assembly and DNA replication genes. The total number of gene types (pangenome) in the seven phages is 176, and 59 of these are unique to individual phages. Their core genomes are much more closely related to one another than to the genome of any other known phage, and they comprise a well-defined cluster within the family Siphoviridae To begin to characterize this group of phages in more experimental detail, we identified the genes that encode the major virion proteins and examined the DNA packaging of the prototypic member, phage 9NA. We show that it uses a pac site-directed headful packaging mechanism that results in virion chromosomes that are circularly permuted and about 13% terminally redundant. We also show that its packaging series initiates with double-stranded DNA cleavages that are scattered across a 170-bp region and that its headful measuring device has a precision of ±1.8%. IMPORTANCE The 9NA-like phages are clearly highly related to each other but are not closely related to any other known phage type. This work describes the genomes of three new 9NA-like phages and the results of experimental analysis of the proteome of the 9NA virion and DNA packaging into the 9NA phage head. There is increasing interest in the biology of phages because of their potential for use as antibacterial agents and for their ecological roles in bacterial communities. 9NA-like phages that infect two bacterial genera have been identified to date, and related phages infecting additional Gram-negative bacterial hosts are likely to be found in the future. This work provides a foundation for the study of these phages, which will facilitate their study and potential use., (Copyright © 2019 American Society for Microbiology.)

This collection of reviews focuses on the most exciting areas of DNA packaging at the current time. Many of the new discoveries are driven by the development of molecular or imaging techniques, and these are providing insights into the complex world of chromatin. As these new techniques continue to improve, we will be able to answer many of the questions we have now, while likely raising many new ones., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD + -dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I- Sce I site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage., Competing Interests: The authors declare no conflict of interest.

Rapid technological advances enabling the construction of designer gene networks, biosynthetic pathways, and even entire genomes are moving the fields of genetics and genomics from descriptive to synthetic applications. Following the synthesis of small viral genomes, advances in DNA assembly and rewriting have enabled the hierarchical synthesis of bacterial genomes, such as Mycoplasma genitalium, as well as recoding of the Escherichia coli genome by reducing the number of codons from 64 to 57. The field has advanced to the point of synthesizing an entire eukaryotic genome. The Synthetic Yeast Genome Project (Sc2.0) is underway and aims to rewrite all 16 Saccharomyces cerevisiae chromosomes by 2018; to date, 6.5 chromosomes have been designed and synthesized. Using bottom-up assembly and applying genome-wide alterations will improve our understanding of genome structure and function. This approach will not only provide a platform for systematic studies of eukaryotic chromosomes but will also generate diverse "streamlined" strains that are potentially suitable for medical and industrial applications. Herein, we review the current state of synthetic genome research and discuss potential applications of this emerging technology., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We compare forces resisting DNA packaging and forces driving DNA ejection in bacteriophage phi29 with theoretical predictions. Ejection of DNA from prohead-motor complexes is triggered by heating complexes after in vitro packaging and force is inferred from the suppression of ejection by applied osmotic pressure. Ejection force from 0% to 80% filling is found to be in quantitative agreement with predictions of a continuum mechanics model that assumes a repulsive DNA-DNA interaction potential based on DNA condensation studies and predicts an inverse-spool conformation. Force resisting DNA packaging from ∼80% to 100% filling inferred from optical tweezers studies is also consistent with the predictions of this model. The striking agreement with these two different measurements suggests that the overall energetics of DNA packaging is well described by the model. However, since electron microscopy studies of phi29 do not reveal a spool conformation, our findings suggest that the spool model overestimates the role of bending rigidity and underestimates the role of intrastrand repulsion. Below ∼80% filling the inferred forces resisting packaging are unexpectedly lower than the inferred ejection forces, suggesting that in this filling range the forces are less accurately determined or strongly temperature dependent.

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or 'procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of 'Headful Packaging' is a DNA-dependent symmetrization of portal protein., Competing Interests: The authors declare no competing financial interests.

Viral terminase enzymes serve as genome packaging motors in many complex double-stranded DNA viruses. The functional motors are multiprotein complexes that translocate viral DNA into a capsid shell, powered by a packaging ATPase, and are among the most powerful molecular motors in nature. Given their essential role in virus development, the structure and function of these biological motors is of considerable interest. Bacteriophage λ-terminase, which serves as a prototypical genome packaging motor, is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. This protomer assembles into a functional higher-order complex that excises a unit length genome from a concatemeric DNA precursor (genome maturation) and concomitantly translocates the duplex into a preformed procapsid shell (genome packaging). While the enzymology of λ-terminase has been well described, the nature of the catalytically competent nucleoprotein intermediates, and the mechanism describing their assembly and activation, is less clear. Here we utilize analytical ultracentrifugation to determine the thermodynamic parameters describing motor assembly and define a minimal thermodynamic linkage model that describes the effects of salt on protomer assembly into a tetrameric complex. Negative stain electron microscopy images reveal a symmetric ring-like complex with a compact stem and four extended arms that exhibit a range of conformational states. Finally, kinetic studies demonstrate that assembly of the ring tetramer is directly linked to activation of the packaging ATPase activity of the motor, thus providing a direct link between structure and function. The implications of these results with respect to the assembly and activation of the functional packaging motor during a productive viral infection are discussed., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1-58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140-162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a "nutcracker" with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Viruses must remain infectious while in harsh extracellular environments. An important aspect of viral particle stability for double-stranded DNA viruses is the energetically unfavorable state of the tightly confined DNA chain within the virus capsid creating pressures of tens of atmospheres. Here, we study the influence of internal genome pressure on the thermal stability of viral particles. Using differential scanning calorimetry to monitor genome loss upon heating, we find that internal pressure destabilizes the virion, resulting in a smaller activation energy barrier to trigger DNA release. These experiments are complemented by plaque assay and electron microscopy measurements to determine the influence of intra-capsid DNA pressure on the rates of viral infectivity loss. At higher temperatures (65-75°C), failure to retain the packaged genome is the dominant mechanism of viral inactivation. Conversely, at lower temperatures (40-55°C), a separate inactivation mechanism dominates, which results in non-infectious particles that still retain their packaged DNA. Most significantly, both mechanisms of infectivity loss are directly influenced by internal DNA pressure, with higher pressure resulting in a more rapid rate of inactivation at all temperatures., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as "molecular staples" at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging., Importance: Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead of procapsids, consequently phage production is affected, indicating the importance of stabilizing interactions during the assembly and maturation reactions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Integration host factor (IHF) is an Escherichia coli protein involved in (i) condensation of the bacterial nucleoid and (ii) regulation of a variety of cellular functions. In its regulatory role, IHF binds to a specific sequence to introduce a strong bend into the DNA; this provides a duplex architecture conducive to the assembly of site-specific nucleoprotein complexes. Alternatively, the protein can bind in a sequence-independent manner that weakly bends and wraps the duplex to promote nucleoid formation. IHF is also required for the development of several viruses, including bacteriophage lambda, where it promotes site-specific assembly of a genome packaging motor required for lytic development. Multiple IHF consensus sequences have been identified within the packaging initiation site (cos), and we here interrogate IHF-cos binding interactions using complementary electrophoretic mobility shift (EMS) and analytical ultracentrifugation (AUC) approaches. IHF recognizes a single consensus sequence within cos (I1) to afford a strongly bent nucleoprotein complex. In contrast, IHF binds weakly but with positive cooperativity to nonspecific DNA to afford an ensemble of complexes with increasing masses and levels of condensation. Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates. At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition. The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.

A prophage vB_CibM-P1 was induced by mitomycin C from the epipelagic strain Citromicrobium bathyomarinum JL354, a member of the alpha-IV subcluster of marine aerobic anoxygenic phototrophic bacteria (AAPB). The induced bacteriophage vB_CibM-P1 had Myoviridae-like morphology and polyhedral heads (approximately capsid 60-100 nm) with tail fibers. The vB_CibM-P1 genome is ~38 kb in size, with 66.0% GC content. The genome contains 58 proposed open reading frames that are involved in integration, DNA packaging, morphogenesis and bacterial lysis. VB_CibM-P1 is a temperate phage that can be directly induced in hosts. In response to mitomycin C induction, virus-like particles can increase to 7 × 10(9) per ml, while host cells decrease an order of magnitude. The vB_CibM-P1 bacteriophage is the first inducible prophage from AAPB.

The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH(-) nucleophile. The ATPase Walker B motif, thus, may be redefined as "β-strand (4-6 hydrophobic-rich amino acids)-DE-hydrophobic/nonpolar amino acid"., (Copyright © 2014 Elsevier Inc. All rights reserved.)

How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle.

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging., (© 2013.)

TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)--the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1). Equally important, it coats and packages the mitochondrial genome. TFAM has been shown to impose a U-turn on LSP DNA; however, whether this distortion is relevant at other sites is unknown. Here we present crystal structures of TFAM bound to HSP1 and to nonspecific DNA. In both, TFAM similarly distorts the DNA into a U-turn. Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not. Moreover, the crystal structures reveal dimerization of DNA-bound TFAM. This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction. We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.

Poxviruses are large, enveloped viruses that replicate in the cytoplasm and encode proteins for DNA replication and gene expression. Hairpin ends link the two strands of the linear, double-stranded DNA genome. Viral proteins involved in DNA synthesis include a 117-kDa polymerase, a helicase-primase, a uracil DNA glycosylase, a processivity factor, a single-stranded DNA-binding protein, a protein kinase, and a DNA ligase. A viral FEN1 family protein participates in double-strand break repair. The DNA is replicated as long concatemers that are resolved by a viral Holliday junction endonuclease.

Over the past decade, nanopores have rapidly emerged as stochastic biosensors. This protocol describes the cloning, expression and purification of the channel of the bacteriophage phi29 DNA-packaging nanomotor and its subsequent incorporation into lipid membranes for single-pore sensing of double-stranded DNA (dsDNA) and chemicals. The membrane-embedded phi29 nanochannel remains functional and structurally intact under a range of conditions. When ions and macromolecules translocate through this nanochannel, reliable fingerprint changes in conductance are observed. Compared with other well-studied biological pores, the phi29 nanochannel has a larger cross-sectional area, which enables the translocation of dsDNA. Furthermore, specific amino acids can be introduced by site-directed mutagenesis within the large cavity of the channel to conjugate receptors that are able to bind specific ligands or analytes for desired applications. The lipid membrane-embedded nanochannel system has immense potential nanotechnological and biomedical applications in bioreactors, environmental sensing, drug monitoring, controlled drug delivery, early disease diagnosis and high-throughput DNA sequencing. The total time required for completing one round of this protocol is around 1 month.

Viral genome packaging into capsids is powered by high-force-generating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient grip-and-release structural change in B form DNA.

Tailed bacteriophages and herpes viruses use powerful ATP-driven molecular motors to translocate their viral genomes into a preformed capsid shell. The bacteriophage T4 motor, a pentamer of the large terminase protein (gp17) assembled at the portal vertex of the prohead, is the fastest and most powerful known, consistent with the need to package a ~170-kb viral genome in approximately 5 min. Although much is known about the mechanism of DNA translocation, very little is known about how ATP modulates motor-DNA interactions. Here, we report single-molecule measurements of the phage T4 gp17 motor by using dual-trap optical tweezers under different conditions of perturbation. Unexpectedly, the motor pauses randomly when ATP is limiting, for an average of 1 s, and then resumes translocation. During pausing, DNA is unpackaged, a phenomenon so far observed only in T4, where some of the packaged DNA is slowly released. We propose that the motor pauses whenever it encounters a subunit in the apo state with the DNA bound weakly and incorrectly. Pausing allows the subunit to capture ATP, whereas unpackaging allows scanning of DNA until a correct registry is established. Thus, the "pause-unpackaging" state is an off-translocation recovery state wherein the motor, sometimes by taking a few steps backward, can bypass the impediments encountered along the translocation path. These results lead to a four-state mechanochemical model that provides insights into the mechanisms of translocation of an intricately branched concatemeric viral genome.

Presented is a detailed kinetic evaluation of the motor component interactions of the DNA translocation ATPase of Bacillus subtilis bacteriophage φ29. The components of the φ29 DNA packaging motor, comprised of both protein and non-protein parts, act in a coordinated manner to translocate DNA into a viral capsid, despite entropically unfavorable conditions. The precise nature of this coordination remains under investigation but recent results have shown that the gp16 pentamer acts to propel the genomic DNA in 10 base pair bursts, implying inter-subunit synchronization. We observe an emergent tandem coordination behavior in the ATPase activity of gp16 as demonstrated by a Hill coefficient of 2.4±0.2, as differentiated from its activity in DNA packaging which has been shown to have a unity Hill coefficient. Due to its relative strength and DNA packaging efficiency, understanding the molecular mechanism of force generation may prove useful to various nanotechnology applications including gene therapy, control of biological ATPases, and the powering of nanoscale mechanical devices., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Despite over 30 years of work, the fundamental structure of eukaryotic chromatin remains controversial. Here, we review the roots of this controversy in disparities between results derived from studies of chromatin in nuclei, chromatin isolated from nuclei, and chromatin reconstituted from defined components. Thanks to recent advances in imaging, modeling, and other approaches, it is now possible to recognize some unifying principles driving chromatin architecture at the level of the ubiquitous '30 nm' chromatin fiber. These suggest that fiber architecture involves both zigzag and bent linker motifs, and that such heteromorphic structures facilitate the observed high packing ratios. Interactions between neighboring fibers in highly compact chromatin lead to extensive interdigitation of nucleosomes and the inability to resolve individual fibers in compact chromatin in situ., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Work from several laboratories over the past 10-15 years has revealed that, within the interphase nucleus, chromosomes are organized into spatially distinct territories [T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292-301 and T. Cremer, M. Cremer, S. Dietzel, S. Muller, I. Solovei, S. Fakan, Chromosome territories-a functional nuclear landscape, Curr. Opin. Cell Biol. 18 (2006) 307-316]. The overall compaction level and intranuclear location varies as a function of gene density for both entire chromosomes [J.A. Croft, J.M. Bridger, S. Boyle, P. Perry, P. Teague,W.A. Bickmore, Differences in the localization and morphology of chromosomes in the human nucleus, J. Cell Biol. 145 (1999) 1119-1131] and specific chromosomal regions [N.L. Mahy, P.E. Perry, S. Gilchrist, R.A. Baldock, W.A. Bickmore, Spatial organization of active and inactive genes and noncoding DNA within chromosome territories, J. Cell Biol. 157 (2002) 579-589] (Fig. 1A, A'). In prophase, when cyclin B activity reaches a high threshold, chromosome condensation occurs followed by Nuclear Envelope Breakdown (NEB) [1]. At this point vertebrate chromosomes appear as compact structures harboring an attachment point for the spindle microtubules physically recognizable as a primary constriction where the two sister chromatids are held together. The transition from an unshaped interphase chromosome to the highly structured mitotic chromosome (compare Figs. 1A and B) has fascinated researchers for several decades now; however a definite picture of how this process is achieved and regulated is not yet in our hands and it will require more investigation to comprehend the complete process. From a biochemical point of view a vertebrate mitotic chromosomes is composed of DNA, histone proteins (60%) and non-histone proteins (40%) [6]. I will discuss below what is known to date on the contribution of these two different classes of proteins and their co-operation in establishing the final mitotic chromosome structure., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The bacteriophage T4 head is an elongated icosahedron packed with 172 kb of linear double-stranded DNA and numerous proteins. The capsid is built from three essential proteins: gp23*, which forms the hexagonal capsid lattice; gp24*, which forms pentamers at 11 of the 12 vertices; and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. Intensive work over more than half a century has led to a deep understanding of the phage T4 head. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as numerous other icosahedral bacteriophages. However, phage T4 displays an unusual membrane and portal initiated assembly of a shape determining self-sufficient scaffolding core. Folding of gp23 requires the assistance of two chaperones, the Escherichia coli chaperone GroEL acting with the phage-coded gp23-specific cochaperone, gp31. The capsid also contains two nonessential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. Through binding to adjacent gp23 subunits, Soc reinforces the capsid structure. Hoc and Soc have been used extensively in bipartite peptide display libraries and to display pathogen antigens, including those from human immunodeficiency virus (HIV), Neisseria meningitides, Bacillus anthracis, and foot and mouth disease virus. The structure of Ip1*, one of a number of multiple (>100) copy proteins packed and injected with DNA from the full head, shows it to be an inhibitor of one specific restriction endonuclease specifically targeting glycosylated hydroxymethyl cytosine DNA. Extensive mutagenesis, combined with atomic structures of the DNA packaging/terminase proteins gp16 and gp17, elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. The cryoelectron microscopy structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at the highest rate known and can package multiple segments. Förster resonance energy transfer-fluorescence correlation spectroscopy studies indicate that DNA gets compressed in the stalled motor and that the terminase-to-portal distance changes during translocation. Current evidence suggests a linear two-component (large terminase plus portal) translocation motor in which electrostatic forces generated by ATP hydrolysis drive DNA translocation by alternating the motor between tensed and relaxed states., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tailed bacteriophages use nanomotors, or molecular machines that convert chemical energy into physical movement of molecules, to insert their double-stranded DNA genomes into virus particles. These viral nanomotors are powered by ATP hydrolysis and pump the DNA into a preformed protein container called a procapsid. As a result, the virions contain very highly compacted chromosomes. Here, I review recent progress in obtaining structural information for virions, procapsids and the individual motor protein components, and discuss single-molecule in vitro packaging reactions, which have yielded important new information about the mechanism by which these powerful molecular machines translocate DNA.

Objective: To assess sperm chromatin integrity in infertile men with varicocele before and after surgical repair., Design: Prospective., Setting: Academic setting., Patient(s): Seventy-two infertile men with varicocele compared with 20 healthy fertile men., Intervention(s): History taking, genital examination, semen analysis, sperm chromatin condensation assessment by aniline blue stain before and 3 months after varicocelectomy., Main Outcome Measure(s): Stained sperm heads (abnormal chromatin condensation) before and 3 months after varicocelectomy., Result(s): The mean percentage of aniline blue-stained sperm heads was significantly higher in infertile men with varicocele compared with fertile controls. The mean percentage of stained sperm heads was significantly decreased in infertile men with varicocele 3 months after surgery compared with the preoperative data. There was a significant negative correlation between percentage of stained sperm heads and normal morphology where nonsignificant correlation was elicited regarding sperm count and sperm motility., Conclusion(s): There is a significant increase of abnormal sperm chromatin condensation in infertile men with varicocele that is markedly improved after varicocelectomy., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Here we explore the use of ethidium to determine relative affinities of different gene delivery vectors for DNA and describe an improved method for studying the interaction. Specifically, we investigate the binding of poly(amidoamine) dendrimers and show that the DNA-dendrimer-ethidium system is far from thermodynamic equilibrium. Moreover, dendrimer surface modification through PEGylation appears to make the interaction with DNA more reversible, which is favorable from the perspective of vector unpacking. Probing the nonequilibrium state of DNA during condensation processes is thus important for developing novel vectors, and further, it could also be useful in the study of chromatin folding.

The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In a prospective randomized fashion, this study evaluated embryo development in vitro after defragmentation versus assisted hatching alone of low grade day 3 embryos. Although a sustained decrease in day 5 fragmentation was observed in the defragmented group versus the assisted hatching only group, no difference in compaction rates or blastula formation rates were appreciated., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Marijuana smokers and animals treated with Δ9-tetrahydrocannabinol, the principal component of marijuana, show alterations of sperm morphology suggesting a role for cannabinoids in sperm differentiation and/or maturation. Because the cannabinoid receptor 1 (CNR1) activation appears to play a pivotal role in spermiogenesis, the developmental stage where DNA is remodeled, we hypothesized that CNR1 receptors might also influence chromatin quality in sperm. We used Cnr1 null mutant (Cnr1-/-) mice to study the possible role of endocannabinoids on sperm chromatin during spermiogenesis. We demonstrated that CNR1 activation regulated chromatin remodeling of spermatids by either increasing Tnp2 levels or enhancing histone displacement. Comparative analysis of wild-type, Cnr1+/-, and Cnr1-/- animals suggested the possible occurrence of haploinsufficiency for Tnp2 turnover control by CNR1, whereas histone displacement was disrupted to a lesser extent. Furthermore, flow cytometry analysis demonstrated that the genetic loss of Cnr1 decreased sperm chromatin quality and was associated with sperm DNA fragmentation. This damage increased during epididymal transit, from caput to cauda. Collectively, our results show that the expression/activity of CNR1 controls the physiological alterations of DNA packaging during spermiogenesis and epididymal transit. Given the deleterious effects of sperm DNA damage on male fertility, we suggest that the reproductive function of marijuana users may also be impaired by deregulation of the endogenous endocannabinoid system.

Biopolymers in vivo are typically subject to spatial restraints, either as a result of molecular crowding in the cellular medium or of direct spatial confinement. DNA in living organisms provides a prototypical example of a confined biopolymer. Confinement prompts a number of biophysics questions. For instance, how can the high level of packing be compatible with the necessity to access and process the genomic material? What mechanisms can be adopted in vivo to avoid the excessive geometrical and topological entanglement of dense phases of biopolymers? These and other fundamental questions have been addressed in recent years by both experimental and theoretical means. A review of the results, particularly of those obtained by numerical studies, is presented here. The review is mostly devoted to DNA packaging inside bacteriophages, which is the best studied example both experimentally and theoretically. Recent selected biophysical studies of the bacterial genome organization and of chromosome segregation in eukaryotes are also covered.

Early preimplantation embryos are sensitive to external osmolarity and use novel mechanisms to accumulate organic osmolytes and thus control their cell volumes and maintain viability. However, these mechanisms are restricted to the cleavage stages of development, and it was unknown whether postcompaction embryos use organic osmolytes. Mouse embryos developing from the 8-cell stage formed blastocoel cavities in vitro at osmolarities up to 360 mOsM. Above this range, several putative organic osmolytes (alanine, glutamine, glycine, and beta-alanine) rescued blastocyst development, but several effective osmoprotectants in cleavage-stage embryos (such as betaine and proline) did not. At physiological osmolarities, each of these compounds resulted in significantly larger blastocysts. This was not due to increased cell numbers, which were unaffected in blastocysts by osmolarity in the range where blastocyst size was rescued by potential organic osmolytes, although cell number was decreased at higher osmolarities and was rescued by each osmolyte. The effective osmolytes were accumulated intracellularly by embryos developing in vitro from the 8-cell stage to blastocysts. However, unlike conventional organic osmolytes in somatic cells or those in cleavage-stage embryos, their intracellular concentrations were not increased with increasing external osmolarity. With the exception of beta-alanine, which is taken up via the beta-amino acid transport system, the effective osmolytes were transported by the B(0,+) system, which becomes highly active in blastocysts. The intracellular accumulation of these osmolytes in postcompaction embryos thus appears to support optimal development and blastocyst expansion at physiological osmolarities and may contribute to the embryo's ability to withstand stress.

Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and lambda phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that approximately 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and 50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the 50-kbp packaged DNA ends are held within 8-9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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.

A common theme in the evolution of sex chromosomes is the massive loss of genes on the sex-specific chromosome (Y or W), leading to a gene imbalance between males (XY) and females (XX) in a male heterogametic species, or between ZZ and ZW in a female heterogametic species. Different mechanisms have evolved to compensate for this difference in dosage of X-borne genes between sexes. In therian mammals, one of the X chromosomes is inactivated, whereas bird dosage compensation is partial and gene-specific. In therian mammals, hallmarks of the inactive X are monoallelic gene expression, late DNA replication and chromatin condensation. Platypuses have five pairs of X chromosomes in females and five X and five Y chromosomes in males. Gene expression analysis suggests a more bird-like partial and gene-specific dosage compensation mechanism. We investigated replication timing and chromosome condensation of three of the five X chromosomes in female platypus. Our data suggest asynchronous replication of X-specific regions on X(1), X(3) and X(5) but show significantly different condensation between homologues for X(3) only, and not for X(1) or X(5). We discuss these results in relation to recent gene expression analysis of X-linked genes, which together give us insights into possible mechanisms of dosage compensation in platypus.

Similar to the assembly of other dsDNA viruses, bacterial virus phi29 uses a motor to translocate its DNA into a procapsid, with the aid of protein gp16 that binds to pRNA 5'/3' helical region. To investigate the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was evaluated as a function of DNA structure (ss- or ds-stranded) or chemistry (purine or pyrimidine). The k(cat) and K(m) in the absence of DNA was 0.016 s(-1) and 351.0 microM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of DNA or pRNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine)>dsDNA>poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involved in the contact with gp16 during DNA packaging.

The packaging of adenovirus (Ad) DNA into virions is dependent upon cis-acting sequences and trans-acting proteins. We studied the involvement of Ad packaging proteins in the serotype specificity of packaging. Both Ad5 and Ad17 IVa2 and L4-22K proteins complemented the growth of Ad5 IVa2 and L4-22K mutant viruses, respectively. In contrast, the Ad5 L1-52/55K protein complemented an Ad5 L1-52/55K mutant virus, but the Ad17 L1-52/55K protein did not. The analysis of chimeric proteins demonstrated that the N-terminal half of the Ad5 L1-52/55K protein mediated this function. Finally, we demonstrate that the L4-33K and L4-22K proteins have distinct functions during infection.

During post-meiotic maturation, male germ cells undergo a formidable reorganization and condensation of their genome. During this phase most histones are globally acetylated and then replaced by sperm-specific basic proteins, named protamines, which compact the genome into a very specific structure within the sperm nucleus. Several studies suggest that this sperm-specific genome packaging structure conveys an important epigenetic message to the embryo. This paper reviews what is known about this fundamental, yet poorly understood, process, which involves not only global changes of the structure of the haploid genome, but also localized specific modifications of particular genomic regions, including pericentric heterochromatin and sex chromosomes. After fertilization, the male genome undergoes a drastic decondensation, and rapidly incorporates new histones. However, it remains different from the maternal genome, bearing specific epigenetic marks, especially in the pericentric heterochromatin region. The functional implications of male post-meiotic and post-fertilization genome reprogramming are not well known, but there is experimental evidence to show that it affects early embryonic development.

During fertilization, spermatozoa contribute genetic and epigenetic factors that affect early embryogenesis. Genetic factors include a haploid genome with intact coding regions and regulatory regions for essential genes. The DNA must contain the proper copy number of essential genes, and cannot have increased single- or double- stranded DNA breaks. Epigenetic factors include a functional centrosome, proper packaging of the chromatin with protamines, modifications of histones, and imprinting of genes. Additionally, the fertilizing spermatozoon provides mRNAs and micro RNAs, which may contribute to the embryonic transcriptome and regulate embryonic gene expression. These epigenetic factors, directly or indirectly, affect the expression of genes in the developing embryo. Each of these contributions represents areas of potential sperm dysfunction, and they are the focus of ongoing research to develop assays which will allow further analysis of their clinical significance. This review briefly describes the current status of research into the genetic and epigenetic contributions of spermatozoa to embryogenesis, and the quest for clinical screening assays. The challenges to validation and clinical application of such testing are also discussed.

During the assembly of many viruses, a powerful molecular motor compacts the genome into a preassembled capsid. Here, we present measurements of viral DNA packaging in bacteriophage phi29 using an improved optical tweezers method that allows DNA translocation to be measured from initiation to completion. This method allowed us to study the previously uncharacterized early stages of packaging and facilitated more accurate measurement of the length of DNA packaged. We measured the motor velocity versus load at near-zero filling and developed a ramped DNA stretching technique that allowed us to measure the velocity versus capsid filling at near-zero load. These measurements reveal that the motor can generate significantly higher velocities and forces than detected previously. Toward the end of packaging, the internal force resisting DNA confinement rises steeply, consistent with the trend predicted by many theoretical models. However, the force rises to a higher magnitude, particularly during the early stages of packaging, than predicted by models that assume coaxial inverse spooling of the DNA. This finding suggests that the DNA is not arranged in that conformation during the early stages of packaging and indicates that internal force is available to drive complete genome ejection in vitro. The maximum force exceeds 100 pN, which is about one-half that predicted to rupture the capsid shell.

An ATP-powered DNA translocation machine encapsidates the viral genome in the large dsDNA bacteriophages. The essential components include the empty shell, prohead, and the packaging enzyme, terminase. During translocation, terminase is docked on the prohead's portal protein. The translocation ATPase and the concatemer-cutting endonuclease reside in terminase. Remarkably, terminases, portal proteins, and shells of tailed bacteriophages and herpes viruses show conserved features. These DNA viruses may have descended from a common ancestor. Terminase's ATPase consists of a classic nucleotide binding fold, most closely resembling that of monomeric helicases. Intriguing models have been proposed for the mechanism of dsDNA translocation, invoking ATP hydrolysis-driven conformational changes of portal or terminase powering DNA motion. Single-molecule studies show that the packaging motor is fast and powerful. Recent advances permit experiments that can critically test the packaging models. The viral genome translocation mechanism is of general interest, given the parallels between terminases, helicases, and other motor proteins.