scholarly journals Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence

2020 ◽  
Vol 48 (20) ◽  
pp. 11602-11614
Author(s):  
Youbin Mo ◽  
Nicholas Keller ◽  
Damian delToro ◽  
Neeti Ananthaswamy ◽  
Stephen C Harvey ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Motor Function ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Viral Genome ◽  
Bacteriophage T4 ◽  
Repeated Measurements ◽  
Genome Packaging ◽  
Sequence Dependence

Abstract Many viruses employ ATP-powered motors during assembly to translocate DNA into procapsid shells. Previous reports raise the question if motor function is modulated by substrate DNA sequence: (i) the phage T4 motor exhibits large translocation rate fluctuations and pauses and slips; (ii) evidence suggests that the phage phi29 motor contacts DNA bases during translocation; and (iii) one theoretical model, the ‘B-A scrunchworm’, predicts that ‘A-philic’ sequences that transition more easily to A-form would alter motor function. Here, we use single-molecule optical tweezers measurements to compare translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor. We observed no significant differences in motor velocities, even with A-philic sequences predicted to show higher translocation rate at high applied force. We also observed no significant changes in motor pausing and only modest changes in slipping. To more generally test for sequence dependence, we conducted correlation analyses across pairs of packaging events. No significant correlations in packaging rate, pausing or slipping versus sequence position were detected across repeated measurements with several different DNA sequences. These studies suggest that viral genome packaging is insensitive to DNA sequence and fluctuations in packaging motor velocity, pausing and slipping are primarily stochastic temporal events.

Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence

2021 ◽  
Author(s):  
Douglas E. Smith ◽  
Youbin E. Mo ◽  
Nick Keller ◽  
Damian delToro ◽  
Neeti Ananthaswamy ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Viral Genome ◽  
Bacteriophage T4 ◽  
Genome Packaging

scholarly journals Function of a Viral Genome Packaging Motor from Bacteriophage T4 is Insensitive to DNA Sequence

2021 ◽  
Vol 120 (3) ◽  
pp. 36a
Author(s):  
Douglas E. Smith ◽  
Youbin Mo ◽  
Nick Keller ◽  
Damian delToro ◽  
Neeti Ananthaswamy ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Viral Genome ◽  
Bacteriophage T4 ◽  
Genome Packaging

scholarly journals A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

2021 ◽  
Author(s):  
Li Dai ◽  
Digvijay Singh ◽  
Suoang Lu ◽  
Vishal Kottadiel ◽  
Reza Vafabakhsh ◽  
...  
Keyword(s):  
Single Molecule ◽  
Viral Genome ◽  
Motor Coordination ◽  
Bacteriophage T4 ◽  
Dna Packaging ◽  
Biological Functions ◽  
Dna Translocation ◽  
Genome Packaging ◽  
Dna Packaging Motor ◽  
Direct Counting

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, by direct counting using single-molecule fluorescence, we have determined 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 count of active/inactive subunit(s), we found, unexpectedly, that the packaging motor can tolerate an inactive sub-unit. However, motors containing an inactive subunit(s) exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a new packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the prevailing notion of strict coordination amongst motor subunits of other packaging motors.

scholarly journals A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Li Dai ◽  
Digvijay Singh ◽  
Suoang Lu ◽  
Vishal I. Kottadiel ◽  
Reza Vafabakhsh ◽  
...  
Keyword(s):  
Single Molecule ◽  
Viral Genome ◽  
Motor Coordination ◽  
Bacteriophage T4 ◽  
Dna Packaging ◽  
Biological Functions ◽  
Dna Translocation ◽  
Single Molecule Fluorescence ◽  
Genome Packaging ◽  
Dna Packaging Motor

AbstractMulti-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.

scholarly journals Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

2019 ◽  
Author(s):  
Alberto Marin-Gonzalez ◽  
Cesar L. Pastrana ◽  
Rebeca Bocanegra ◽  
Alejandro Martín-González ◽  
J.G. Vilhena ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mechanical Response ◽  
Magnetic Tweezers ◽  
Crystallographic Structure ◽  
Chain Model ◽  
Microscopy Imaging ◽  
Length Scales

ABSTRACTA-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, their response to forces remains unknown and the variability of their flexibility reported for different length scales has precluded a comprehensive description of the mechanical properties of these molecules. Here, we rationalize the mechanical properties of A-tracts across multiple length scales using a combination of single-molecule experiments and theoretical polymer models applied to DNA sequences present in the C. elegans genome. Atomic Force Microscopy imaging shows that phased A-tracts induce long-range (∼200 nm) bending. Moreover, the enhanced bending originates from an intrinsically bent structure rather than as a consequence of larger flexibility. In support of this, our data were well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments confirm that the observed bent is intrinsic to the sequence and does not rely on particular ionic conditions. Using optical tweezers, we assess the local rigidity of A-tracts at high forces and unravel an unusually stiff character of these sequences, as quantified by their large stretch modulus. Our work rationalizes the complex multiscale flexibility of A-tracts, shedding light on the cryptic character of these sequences.

scholarly journals DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA.

1996 ◽  
Vol 16 (7) ◽  
pp. 3773-3780 ◽  
Author(s):  
S D Liang ◽  
R Marmorstein ◽  
S C Harrison ◽  
M Ptashne
Keyword(s):  
Crystal Structure ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Tight Binding ◽  
Recognition Sequence ◽  
Sequence Dependence ◽  
Network Of Hydrogen Bonds ◽  
Dna Binding Assay ◽  
Related Proteins

Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences. We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay. For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure. Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent. The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds. Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site. PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence. The identities of the internal 6 bp do not influence the binding of PPR1. We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5' to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1. Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1.

scholarly journals CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Francisco de Asis Balaguer ◽  
Clara Aicart-Ramos ◽  
Gemma LM Fisher ◽  
Sara de Bragança ◽  
Eva M Martin-Cuevas ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Specific Interaction ◽  
Magnetic Tweezers ◽  
One Dimensional ◽  
Sliding Clamp ◽  
Dimensional Diffusion ◽  
Absolute Requirement ◽  
Bacterial Chromosomes

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPgS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum-dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.

scholarly journals DNA sequence encodes the position of DNA supercoils

2017 ◽  
Author(s):  
Sung Hyun Kim ◽  
Mahipal Ganji ◽  
Jaco van der Torre ◽  
Elio Abbondanzieri ◽  
Cees Dekker
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Dna Sequences ◽  
Structural Information ◽  
Three Dimensional ◽  
Decisive Role ◽  
Dimensional Structure ◽  
Transcription Start Sites ◽  
Cellular Processes

AbstractThe three-dimensional structure of DNA is increasingly understood to play a decisive role in gene regulation and other vital cellular processes, which has triggered an explosive growth of research on the spatial architecture of the genome. Many studies focus on the role of various DNA-packaging proteins, crowding, and confinement in organizing chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we use a fluorescence-based single-molecule technique to visualize plectonemes, the extended intertwined DNA loops that form upon twisting DNA. Remarkably, we find that the underlying DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific positions. We explore a variety of DNA sequences to determine what features influence pinning, and we develop a physical model that predicts the level of plectoneme pinning in excellent agreement with the data. The intrinsic curvature measured over a range of ~70 base pairs is found to be the key property governing the supercoiled structure of DNA. Our model predicts that plectonemes are likely to localize directly upstream of prokaryotic transcription start sites, and this prediction is experimentally verifiedin vitro.Our results reveal a hidden code in DNA that helps to spatially organize the genome.

scholarly journals Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

2020 ◽  
Vol 48 (9) ◽  
pp. 5024-5036
Author(s):  
Alberto Marin-Gonzalez ◽  
Cesar L Pastrana ◽  
Rebeca Bocanegra ◽  
Alejandro Martín-González ◽  
J G Vilhena ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mechanical Response ◽  
Magnetic Tweezers ◽  
Crystallographic Structure ◽  
Chain Model ◽  
Microscopy Imaging ◽  
Wide Range

Abstract A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

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