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Dive into the research topics where John F. Marko is active.

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Featured researches published by John F. Marko.


Current Biology | 1997

Interphase chromosomes undergo constrained diffusional motion in living cells

Wallace F. Marshall; Aaron F. Straight; John F. Marko; Jason R. Swedlow; Abby F. Dernburg; Andrew S. Belmont; Andrew W. Murray; David A. Agard; John W. Sedat

BACKGROUND Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus. RESULTS To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture. CONCLUSIONS We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.


The EMBO Journal | 2000

One- and three-dimensional pathways for proteins to reach specific DNA sites

Neil P. Stanford; Mark D. Szczelkun; John F. Marko; Stephen E. Halford

Proteins that interact with specific DNA sites bind to DNA at random and then translocate to the target site. This may occur by one‐dimensional diffusion along the DNA, or through three‐dimensional space via multiple dissociation/re‐associations. To distinguish these routes, reactions of the EcoRV endonuclease were studied on substrates with two EcoRV sites separated by varied distances. The fraction of encounters between the DNA and the protein that resulted in the processive cleavage of both sites decreased as the length of intervening DNA was increased, but not in the manner demanded for one‐dimensional diffusion. The variation in processivity with inter‐site spacing shows instead that protein moves from one site to another through three‐dimensional space, by successive dissociation/re‐associations, though each re‐association to a new site is followed by a search of the DNA immediately adjacent to that site. Although DNA‐binding proteins are usually thought to find their target sites by one‐dimensional pathways, three‐dimensional routes may be more common than previously anticipated.


Nucleic Acids Research | 2012

Self-organization of domain structures by DNA-loop-extruding enzymes

Elnaz Alipour; John F. Marko

The long chromosomal DNAs of cells are organized into loop domains much larger in size than individual DNA-binding enzymes, presenting the question of how formation of such structures is controlled. We present a model for generation of defined chromosomal loops, based on molecular machines consisting of two coupled and oppositely directed motile elements which extrude loops from the double helix along which they translocate, while excluding one another sterically. If these machines do not dissociate from DNA (infinite processivity), a disordered, exponential steady-state distribution of small loops is obtained. However, if dissociation and rebinding of the machines occurs at a finite rate (finite processivity), the steady state qualitatively changes to a highly ordered ‘stacked’ configuration with suppressed fluctuations, organizing a single large, stable loop domain anchored by several machines. The size of the resulting domain can be simply regulated by boundary elements, which halt the progress of the extrusion machines. Possible realizations of these types of molecular machines are discussed, with a major focus on structural maintenance of chromosome complexes and also with discussion of type I restriction enzymes. This mechanism could explain the geometrically uniform folding of eukaryote mitotic chromosomes, through extrusion of pre-programmed loops and concomitant chromosome compaction.


Proceedings of the National Academy of Sciences of the United States of America | 2002

Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold

Michael G. Poirier; John F. Marko

Isolated newt (Notophthalmus viridescens) chromosomes were studied by using micromechanical force measurement during nuclease digestion. Micrococcal nuclease and short-recognition-sequence blunt-cutting restriction enzymes first remove the native elastic response of, and then to go on to completely disintegrate, single metaphase newt chromosomes. These experiments rule out the possibility that the mitotic chromosome is based on a mechanically contiguous internal non-DNA (e.g., protein) “scaffold”; instead, the mechanical integrity of the metaphase chromosome is due to chromatin itself. Blunt-cutting restriction enzymes with longer recognition sequences only partially disassemble mitotic chromosomes and indicate that chromatin in metaphase chromosomes is constrained by isolated chromatin-crosslinking elements spaced by ≈15 kb.


Proceedings of the National Academy of Sciences of the United States of America | 2001

Force and kinetic barriers to unzipping of the DNA double helix

Simona Cocco; Rémi Monasson; John F. Marko

A theory of the unzipping of double-stranded (ds) DNA is presented, and is compared to recent micromanipulation experiments. It is shown that the interactions which stabilize the double helix and the elastic rigidity of single strands (ss) simply determine the sequence dependent =12 pN force threshold for DNA strand separation. Using a semi-microscopic model of the binding between nucleotide strands, we show that the greater rigidity of the strands when formed into dsDNA, relative to that of isolated strands, gives rise to a potential barrier to unzipping. The effects of this barrier are derived analytically. The force to keep the extremities of the molecule at a fixed distance, the kinetic rates for strand unpairing at fixed applied force, and the rupture force as a function of loading rate are calculated. The dependence of the kinetics and of the rupture force on molecule length is also analyzed.A theory of the unzipping of double-stranded DNA is presented and is compared to recent micromanipulation experiments. It is shown that the interactions that stabilize the double helix and the elastic rigidity of single strands simply determine the sequence-dependent ≈12-pN force threshold for DNA strand separation. Using a semimicroscopic model of the binding between nucleotide strands, we show that the greater rigidity of the strands when formed into double-stranded DNA, relative to that of isolated strands, gives rise to a potential barrier to unzipping. The effects of this barrier are derived analytically. The force to keep the extremities of the molecule at a fixed distance, the kinetic rates for strand unpairing at fixed applied force, and the rupture force as a function of loading rate are calculated. The dependence of the kinetics and of the rupture force on molecule length is also analyzed.


Nature | 1999

A kinetic proofreading mechanism for disentanglement of DNA by topoisomerases

Jie Yan; Marcelo O. Magnasco; John F. Marko

Cells must remove all entanglements between their replicated chromosomal DNAs to segregate them during cell division. Entanglement removal is done by ATP-driven enzymes that pass DNA strands through one another, called type II topoisomerases. In vitro, some type II topoisomerases can reduce entanglements much more than expected, given the assumption that they pass DNA segments through one another in a random way. These type II topoisomerases (of less than 10 nm in diameter) thus use ATP hydrolysis to sense and remove entanglements spread along flexible DNA strands of up to 3,000 nm long. Here we propose a mechanism for this, based on the higher rate of collisions along entangled DNA strands, relative to collision rates on disentangled DNA strands. We show theoretically that if a type II topoisomerase requires an initial ‘activating’ collision before a second strand-passing collision, the probability of entanglement may be reduced to experimentally observed levels. This proposed two-collision reaction is similar to ‘kinetic proofreading’ models of molecular recognition.


Nucleic Acids Research | 2011

Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA

John S. Graham; Reid C. Johnson; John F. Marko

The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of kexch = 6 × 104 M−1s−1. The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation.


Molecular Microbiology | 2012

Variation of the folding and dynamics of the Escherichia coli chromosome with growth conditions

Nastaran Hadizadeh Yazdi; Calin C. Guet; Reid C. Johnson; John F. Marko

We examine whether the Escherichia coli chromosome is folded into a self‐adherent nucleoprotein complex, or alternately is a confined but otherwise unconstrained self‐avoiding polymer. We address this through in vivo visualization, using an inducible GFP fusion to the nucleoid‐associated protein Fis to non‐specifically decorate the entire chromosome. For a range of different growth conditions, the chromosome is a compact structure that does not fill the volume of the cell, and which moves from the new pole to the cell centre. During rapid growth, chromosome segregation occurs well before cell division, with daughter chromosomes coupled by a thin inter‐daughter filament before complete segregation, whereas during slow growth chromosomes stay adjacent until cell division occurs. Image correlation analysis indicates that sub‐nucleoid structure is stable on a 1 min timescale, comparable to the timescale for redistribution time measured for GFP–Fis after photobleaching. Optical deconvolution and writhe calculation analysis indicate that the nucleoid has a large‐scale coiled organization rather than being an amorphous mass. Our observations are consistent with the chromosome having a self‐adherent filament organization.


Chromosome Research | 2008

Micromechanical studies of mitotic chromosomes

John F. Marko

Mitotic chromosomes respond elastically to forces in the nanonewton range, a property important to transduction of stresses used as mechanical regulatory signals during cell division. In addition to being important biologically, chromosome elasticity can be used as a tool for investigating the folding of chromatin. This paper reviews experiments studying stretching and bending stiffness of mitotic chromosomes, plus experiments where changes in chromosome elasticity resulting from chemical and enzyme treatments were used to analyse connectivity of chromatin inside chromosomes. Experiments with nucleases indicate that non-DNA elements constraining mitotic chromatin must be isolated from one another, leading to the conclusion that mitotic chromosomes have a chromatin ‘network’ or ‘gel’ organization, with stretches of chromatin strung between ‘crosslinking’ points. The as-yet unresolved questions of the identities of the putative chromatin crosslinkers and their organization inside mitotic chromosomes are discussed.


Biophysical Journal | 1997

Driving proteins off DNA using applied tension

John F. Marko; Eric D. Siggia

Proteins that bind DNA so as to reduce its end-to-end length can be dissociated by application of force. The thermodynamics of this process are discussed, with special attention to the case of histones bound to DNA (i.e., a string of nucleosomes, or chromatin fiber). The histone octamer is predicted to be driven off chromatin fiber for tensions >2 piconewtons.

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Jie Yan

National University of Singapore

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Mingxuan Sun

Northwestern University

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Andrew D. Stephens

University of North Carolina at Chapel Hill

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Botao Xiao

Northwestern University

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Dunja Skoko

University of Illinois at Chicago

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Simona Cocco

École Normale Supérieure

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