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Is DNA mutation locally energetically stabilizing the DNA molecule

Is DNA mutation locally energetically stabilizing the DNA molecule


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I am no biologist, but as a physicist, a spontaneous mutation (seen as a chemical transformation) should lower the energy of the system, at least locally. So I wonder if any research has been done along these lines for the DNA.


a spontaneous mutation (seen as a chemical transformation) should lower the energy of the system

Why do you think that? Because it's an endothermic reaction?

Consider that mutations don't happen “just like that” (at least to my knowledge) -they are triggered by external energy influx from things like reactive oxygen species or ionising radiation. So yes, they cost energy but that energy doesn't come energetically unstable chemical bonds; it is supplemented from external sources.

So it's not necessarily true that mutations produce lower-energy chemical bonds.


I'm going to attempt an answer to this at the level of undergraduate biochemistry: apologies in advance if this is not sufficiently sophisticated.

The most frequent spontaneous mutation is deamination of cytosine to uracil (hydrolysis with loss of ammonia). Strictly in terms of the double helical structure of the DNA molecule this, it seems to me, would be destabilizing because of the change from a GC base pair (3 hydrogen bonds) to a GU base pair (2 hydrogen bonds).


The reaction kinetics of DNA mutations are complicated by the fact that the DNA doesn't exist in isolation, and in fact is actively processed and maintained by the cellular machinery.

For example, as Alan Boyd notes, the most common spontaneous mutation is the deamination of cytosine to uracil: Cyt + H2O → NH3 + Ura. On its own, the reverse of this reaction, i.e. spontaneous amination of uracil to cytosine, would be very unlikely, if only because the low concentration of ammonia compared to water in cellular fluid.

However, in a living cell, there are DNA repair enzymes that actively look for uracil in DNA and remove it, allowing other enzymes to come along and replace it with the original cytosine. This keeps the effective Cyt → Ura mutation rate far below what it would be in the absence of active repair.


'Jumping genes' help stabilize DNA folding patterns

"Jumping genes"—bits of DNA that can move from one spot in the genome to another—are well-known for increasing genetic diversity over the long course of evolution. Now, new research at Washington University School of Medicine in St. Louis indicates that such genes, also called transposable elements, play another, more surprising role: stabilizing the 3-D folding patterns of the DNA molecule inside the cell's nucleus.

The study appears Jan. 24 in the journal Genome Biology.

The DNA molecule inside the nucleus of any human cell is more than six feet long. To fit into such a small space, it must fold into precise loops that also govern how genes are turned on or off. It might seem counterintuitive that bits of DNA that randomly move about the genome can provide stability to these folding patterns. Indeed, the discovery contradicts a long-held assumption that the precise order of letters in the DNA sequence always dictates the broader structure of the DNA molecule.

"In places where the larger 3-D folding of the genome is the same between mice and humans, you expect the sequence of the letters of the DNA anchoring that shape to be conserved there as well," said senior author Ting Wang, Ph.D., the Sanford C. and Karen P. Loewentheil Distinguished Professor of Medicine. "But that's not what we found, at least not in the portions of the genome that in the past have been called 'junk DNA.'"

Studying DNA folding in mouse and human blood cells, the researchers found that in many regions where the folding patterns of DNA are conserved through evolution, the genetic sequence of the DNA letters establishing these folds is not. It is ever so slightly displaced. But this changing sequence, a genetic turnover, doesn't cause problems. Because the structure largely stays the same, the function presumably does, too, so nothing of importance changes.

"We were surprised to find that some young transposable elements serve to maintain old structures," said first author Mayank N.K. Choudhary, a doctoral student in Wang's lab. "The specific sequence may be different, but the function stays the same. And we see that this has happened multiple times over the past 80 million years, when the common ancestors of mice and humans first diverged from one another."

The fact that a new transposable element can insert itself and serve the same role as an existing anchor creates a redundancy in the regulatory portions of the genome—regions of the DNA molecule that determine how and when genes are turned on or off.

According to the researchers, this redundancy makes the genome more resilient. In providing both novelty and stability, jumping genes may help the mammalian genome strike a vital balance—allowing animals the flexibility to adapt to a changing climate, for example, while preserving biological functions required for life, protecting against the DNA damage that is wrought by living and reproducing on Earth over the span of deep time, measured in tens to hundreds of millions of years.

Even so, the researchers were careful to distinguish between portions of the genome that hold genes responsible for producing proteins and the rest of the genome. In genes that code for proteins, the genetic sequence and the structure are both conserved, and this study does not contradict that. However, the new research suggests that jumping genes in the non-protein coding areas of the genome follow different rules of conservation than the protein-coding genes.

"Our study changes how we interpret genetic variation in the noncoding regions of the DNA," Wang said. "For example, large surveys of genomes from many people have identified a lot of variations in noncoding regions that don't seem to have any effect on gene regulation, which has been puzzling. But it makes more sense in light of our new understanding of transposable elements—while the local sequence can change, but the function stays the same.

"We may need to revisit these types of studies in light of the new understanding we now have of transposable elements," he added. "We have uncovered another layer of complexity in the genome sequence that was not known before."


MATERIALS AND METHODS

Expression and purification of HMO1 protein

Plasmid pJ1870 encoding yeast HMO1 cloned in-frame with an N-terminal His6 tag in expression vector pTEV derived from pET15b (Novagen) was transformed into Escherichia coli BL21(DE3) (Agilent Technologies), and grown in 250-ml LB culture at 37°C with shaking until the culture reached a cell density corresponding to an OD600 of 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and cells were grown at 37°C overnight with shaking, pelleted by centrifugation at 6000 g, and the cell pellet was then resuspended in 10 ml binding buffer (50 mM NaPO4, 300 mM NaCl, pH 7.5) containing 10 mM phenylmethlysulfonylfluoride (PMSF) and passed five times through an Emulsiflex C-5 high-pressure homogenizer (Avestin). The lysate was clarified by centrifugation at 22 000 g for 45 min at 4°C and the supernatant recovered. His6-tagged protein was purified using Ni-NTA agarose resin (Qiagen) per the manufacturer's recommendations. Briefly, washed Ni-NTA agarose resin was added in a 1:4 (v:v) ratio to the lysate, gently rotated at 4°C for 1 h, then loaded onto a 1.5 × 15 cm column. Resin-bound protein was washed with 200 ml wash buffer (50 mM NaPO4, 300 mM NaCl, 20 mM imidazole, pH 7.5). These conditions were sufficient to release bound contaminating DNA. Protein was eluted from the resin with elution buffer (50 mM NaPO4, 300 mM NaCl, 250 mM imidazole, pH 7.5) collecting 2 ml fractions until no protein was detectable with Bradford reagent. Fractions containing the protein of interest were combined and reduced to 2 ml using centrifugal cartridges (Vivaspin), and proteins were dialyzed at 4°C against 1 liter buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT), followed by a second dialysis against the same buffer containing 5% glycerol.

Purified protein was detagged using His6-tagged-TEV protease followed by dialysis into binding buffer to remove residual imidazole. Detagged protein was purified from the His6-tag and His6-tagged-TEV protease by column chromatography as described above, but with elution using nine steps of increasing imidazole concentration between 5 and 250 mM. Fractions with the desired protein were combined and reduced to 2 ml by centrifugal concentration and proteins were dialyzed at 4°C against 1 liter buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 1 mM EDTA and 1 mM DTT, followed by a second dialysis against the same buffer containing 5% glycerol. Protein quality was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, DNA affinity quantitated by electrophoretic gel mobility shifts assays and DNA bending confirmed by enhancement of T4 DNA ligase-mediated DNA cyclization.

Protein–DNA sample preparation for atomic force microscopy

We use 4361 bp linearized plasmid DNA pBR322. Linearization was performed by PvuII digestion followed by phenol extraction ( 20). The freshly cleaved mica surface was exposed to 5 mM Mg 2+ for 20 min at ambient temperature and pressure. The surface was then rinsed with distilled water and air dried. A DNA solution of 0.11 nM was deposited and allowed to incubate for 30 min, then rinsed and dried with argon gas. In order to image the protein-bound DNA complexes, 3 nM protein was incubated with 0.11 nM linearized plasmid pBR322 DNA with 10 mM Tris–HCl (pH 8.0) and 5 mM Mg 2+ , then deposited on the mica surface, left to equilibrate for 20 min, and finally rinsed and dried with argon gas.

Optical tweezers

To investigate and characterize HMO1–DNA interactions, we use dual beam optical tweezers with 830 nm lasers, which can sustain a force up to 300 pN. The experimental setup consists of focusing two laser beams into a 1 μm diameter spot. A bead of high refractive index compared to the surrounding medium will be attracted to the focal point. A second bead is immobilized by a glass micropipette. A high resolution piezoelectric stage (0.15 nm resolution, Npoint) is used to extend the DNA. The force is measured from the refraction of the laser from the polystyrene bead. Once the single molecule is captured between the two beads, the solution around the DNA is exchanged by flowing in 10 times the flow cell volume of protein solution at fixed concentration followed by thermal equilibration and DNA stretching. The buffer solution consisted of 100 mM Na + and 10 mM HEPES pH 7.5. Bridging and looping effects were observed by holding DNA molecules at stretching forces <1 pN.

Here Θ is the DNA fractional site occupancy, KD is the dissociation constant, n is the binding site size (n = 26 bp), c is the concentration and ω is binding cooperativity parameter.

Atomic force microscopy imaging

A Bruker Nanoscope V MultiMode 8 atomic force microscope is used with Peak-Force Tapping™ mode. In this mode, a force curve is obtained at every pixel of the image. The peak force is used as a feedback parameter in order to image topography. The sample can be scanned at lower forces and with shorter contact time, thus protecting delicate samples. For imaging in air, a silicon cantilever is used (resonance frequency = 70 kHz, spring constant = 0.4 N/m and tip radius = 2 nm). The experiments were performed at room temperature. Images are processed using Nanoscope Analysis software, which consists of subtracting the average of each line in order to remove planar artifacts. The scan range used was 1 μm × 1 μm and 2 μm × 2 μm at 512 × 512 pixels and at 1024 × 1024 pixels, respectively. To quantify the atomic force microscopy (AFM) images, DNA molecules were traced and analyzed with NCTracer, software developed by the Neurogeometry Lab at Northeastern University ( 22, 23).


Chapter 4 - Samenvatting Molecular Biology of the Cell

A deoxyribonucleic acid consists of two long polynucleotide chains composed of four types of nucleotide subunits. The chains can also be called DNA strands. The chains run antiparallel to each other, and hydrogen bonds between the base portions of the nucleotides hold the two chains together.

Nucleotide are composed of a five-carbon sugar to which are attaced one or more phosphate groups and a nitrogen-containing base. The nucleotides are covalently linked together in a chain through the sugars and phosphates, which thus form a backbone of alternating sugar-phosphate.

The linking of the nucleotides gives the DNA strand a chemical polarity. The two ends of the chain are easily distinguishable, as one has a hole (3’ hydroxyl) and the other a knob (5’ phosphate).

Because the chains are held together by hydrogen-boning between the bases, the bases are on the inside of the double helix. The complementary base-pairing enables the base pairs to be packed in the energetically most favorable arrangement.

The structure of DNA provides a mechanism for heredity

Each strand can act as a template for producing a complementary strand, this enables the cell to copy, or replicate, its genome.

Genome – complete store of information in an organism’s DNA

In eukaryotes, DNA is enclosed in a cell nucleus

All the DNA in a eukaryotic cell is sequestered in a nucleus. This compartment is delimited by a nuclear envelope formed by two concentric lipid bilayer membranes. These membranes are punctured at intervals by large nuclear pores, through which molecules move between the nucleus and the cytosol.

The nuclear envelope is directly connected to the extensive system of intracellular membranes called the endoplasmatic reticulum. And it is mechanically supported by a network of intermediate filaments called the nuclear lamina.

Chromosomal DNA and its packaging in the chromatin fiber

Eukaryotic DNA is packaged into a set of chromosomes

Each chromosome consists of a single, long linear DNA molecule along with the proteins that fold and pack the DNA thread into a compact structure. The complex of DNA and tightly bound protein is called chromatin.

Bacteria lack a special nuclear compartment, and they carry their genes on a single DNA molecule, which is often circular.

Homologous chromosomes (homologs) are the maternal and paternal chromosomes of a pair. The only nonhomologous pairs are the sex chromosomes in males.

Chromosomes contain long strings of genes

Because of differences in the amount of noncoding DNA, the genomes closely related organisms can vary several hundredfold in their DNA content, even though they contain roughly the same number of genes.

There is no simple relationship between chromosome number, complexity of the organism, and the total genome.

The nucleotide sequence of the human genome shows how our genes are arranged

Nearly half of the chromosomal DNA is made up of mobile pieces of DNA that have gradually inserted themselves in the chromosomes over evolutionary time, transposable elements.

The coding sequences are called exons, the intervening (non-coding) sequences are introns.

Each gene is associated with regulatory DNA sequences, which are responsible for ensuring that the gene is turned on or off at the proper time.

Each DNA molecule that forms a linear chromosome must contain a centromere, two telomeres, and replication origins

Cell cycle – provides for a temporal separation between the duplication of chromosomes and their segregation into two daughter cells. During interphase genes are expressed and chromosomes are replicated, with the two replicas remaining together as a pair of sister chromatids. The highly condensed chromosomes in a dividing cell are known as mitotic chromosomes.

DNA replication origin – the location at which duplication of the DNA begins

Centromere – allows one copy of each duplicated and condensed chromosome to be pulled into each daughter cell when a cell divides.

Kinechore – protein complex that forms at the centromere and attached the duplicated chromosomes to the mitotic spindle

Telomeres – ends of chromosome, contain repeated nuceleotide sequences that enable the ends of chromosomes to be efficiently replicated. And together with the regions adjoining

A lot of lysine and arginine in the core histones, their positive charges can effectively neutralize the negatively charged DNA backbone.

Each of the core histones has a N-terminal amino acid ‘tail’, which extends out from the DNA-histone core. These tails are subject to several types of covalent modification that in turn control critical aspects of chromatin structure and function.

Nucleosomes have a dynamic structure, and are frequentlu subjected to changes catalyzed by ATP-dependent chromatin remodeling complexes

For the chromatin in a cell, loosening of DNA-histone contacts is required, because euakryotic cells contain a large variety of ATP-dependent chromatin remodeling complexes. These complexes include a subunit that hydrolyze ATP. This subunit binds both to the protein core of the nucleosome and to the double-stranded DNA that winds around it. By using the energy of ATP hydrolysis to move this DNA relative to the core, the protein complex changes the structure, making the DNA less tighly bound to the histone core.

Through repeated cycles of ATP hydrolysis that pull the nucleosome core aong the DNA helix, the remodeling complexes can catalyze nucleosome sliding. They can reposition nucleosomes to expose specific regions of DNA.

By cooperating with histone chaperones, some remodeling complexes are able to remove either all or part of the nucleosome core from a nucleosome – catalyzing either an exchange of its H2A-H2B histones, or the complete removal of the octameric core.

Nucleosomes are usually packed together into a compact chromatin fiber

The nucleosomes are packed on top of one another, generating arrays in which the DNA is even more highly condensed.

Nucleosome-to-nucleosome linkages that involve histone tails, most notable H4 tails, constitute one important factor on what causes nucleosomes to stack so tighly on each other. Another important factor is an additional histone that is often present in a 1-to-1 ration with nucleosome cores, known as histone H1. This linker histone is larger that individual core histones and it has been considerably less well conserved during evolution.

A single H1 molecule binds to each nucleosome, connectiong DNA and protein, and changing the path of the DNA as it exits from the nucleosome. This change is thought to help compact nucleosomal DNA.

Chromatin structure and function

Heterochromatin is highly organized and restricts gene expression

Heterochromatin – highly condensed, euchromatin – less condensed.

The DNA in heterochromatin contains few genes, and when euchromatin regions are converted to a heterchromatic state, their genes are generally switched off as a result.

Heterochromatin should not be thought of a simply encapsulating ‘dead’DNA, but rather as a descriptor for compact chromatin domains that share the common feature of being unusually resistant to gene expression.

The heterochromatic state is self-propagating

A piece of chromosome that is normally euchromatic can be translocated into the neighborhood of heterochromatin, this often causes silencing – inactivation – of the active genes. This phenomenon is referred to as a position effect.

In chromosome breakage-and-rejoining events of the zone of silinceing, where euchromatin is converted to a heterochromatic state, spreads for different distances in different early cells in the embryo. Remarkably, these differences then are perpetuated for the rest of the

A similar process is used to remove histone modifications from specific regions, in this case an “eraser enzyme” is recruited to the complex

Barrier DNA sequences block the spread of reader-writer complexes and thereby separate neighboring chromatin domains

Certain DNA sequences mark the boundaries of chromatin domains and separate one such domain from another.

The chromatin in centromeres reveals how histone variants can create special structures

In many complex organisms each centromere is embedded in a stretch of special centromeric chromatin that persists throughout interphase, even though the centromere- mediated attachment to the spindle and movement of DNA occur only during mitosis.

Experiments with frog embryos suggest that both activating and repressive chromatin structures can be inherited epigenetically

Epigenetic inheritance plays a central part in the creation of multicellular organisms. Their differentiated cell types become established during development, and persist thereafter even though repeated cell-division

Chromatin structures are important for eukaryotic chromosome function

To form a complex multicellular organism, the cells in different lineages must specialize by changing the accessibility and activity of many hundreds of genes.

The global structure of chromosomes

Chromosomes are folded into large loops of chromatin

Polytene chromosomes are uniquely useful for visualizing chromatin structures

Genome comparisons reveal functional DNA sequences by their conservation throughout evolution

Conserved regions – closely similar pieces of DNA sequences. In addition to revealing those DNA sequences that encode functionally important exons and RNA molecules, these conserved regions will include regulatory DNA sequences as well as DNA sequences with functions that are not yet knowm. Most nonconserved regions will reflect DNA whose sequence is much less likely to be critical for function.

Genome alterations are caused by failures of the normal mechanics for copying and maintaining DNA, as well as by transposable DNA elements

Most od the genetic changes that occur result simply form failures in the normal mechanics by which genomes are copied or repaired when damaged, although the movement of transposable DNA element also plays an important part.

Error is DNA replication, recombination or repair lead to either simple local changes (point mutations) or to large-scale genome rearrangements (like deletions, duplications, inversions, and translocations of DNA from one to the other chromosome.

Genomes contain mobile DNA elements that are an important source of genomic change. These transposons are parasitic DNA sequences that can spread within the genomes they colonize. In the process, they often disrupt the function or alter the regulation of existing genes.

The genome sequences of two species differ in proportion to the length of time since they have separately evolved

The basic organizing framework for comparative genomics is the phylogentic tree.

Purifying selection – selection that eliminates individuals carrying mutations that interfere with important genetic functions

Phylogenetic trees constructed from a comparison of DNA sequences trace the relationships of all organisms

We can made a molecular clock for evolution. The clocks runs most rapidly in sequences that are not subject to purifying selection. The clock runs most slowly for sequences that are subject to strong functional constraints.

The pace at which molecular clocks run is determined not only by the degree of purifying selection, but also by the mutation rate.

A comparison of human and mouse chromosomes shows how the structures of genomes diverge

As would be expected, the human and chimpanzee genomes are much more alike than are the human and mouse genomes, even though all three genomes are roughly the same size and contain nearly identical sets of genes. While the way that the genome is organized into chromosomes is almost identical between humans and chimpanzees, this organization has diverged greatly between humans and mice. An unexpected conclusion from a detailed comparison of the complete mouse and human genome sequences, confirmed by subsequent comparisons between the genomes of other vertebrates, is that small blocks of DNA sequence are being deleted from and added to genomes at a surprisingly rapid rate. Thus, if we assume that our common ancestor had a genome of human size (about 3.2 billion nucleotide pairs), mice would have lost a total of about 45% of that genome from accumulated deletions during the past 80 million years, while humans would have lost about 25%.

The size of a vertebrate genome reflects the relative rates of DNA addition and DNA loss in a lineage

While initially a mystery, we now have a simple explanation for such large differences in genome size between similar organisms: because all vertebrates experience a continuous process of DNA loss and DNA addition, the size of a genome merely depends on the balance between these opposing processes acting over millions of years.

We can infer the sequence of some ancient genomes

Multispecies sequence comparisons identify conserved DNA sequences if unknown function

Many of the conserved sequences that do not code for proteins are known to produce untranslated RNA molecules.

Changes in previously conserved sequences can help decipher critical steps in evolution


How an Enzyme Repairs DNA via a “Pinch-Push-Pull” Mechanism

Thymine DNA glycosylase (TDG) in complex with DNA is shown on the top left panel. Free energy profile for the TDG-induced base flipping is shown in the top right panel. Structures of the flipped out DNA base interacting the enzyme at various intermediate states are shown and mapped onto the base flipping path (as indicated by arrows).  Credit: Dr. Chunli Yan and Tom Dodd, Georgia State University

At first, the alliterative phrase sounds remarkably simplistic: “pinch-push-pull.”

But when it comes to preserving one of biology’s basic processes – the replication of the genetic code – this single-syllabic triplet spells the difference between normal cell division and a potentially hazardous mutation.

In a study published in the May 21, 2018 issue of the Proceedings of the National Academy of Sciences, a team of researchers – aided with supercomputing resources from the San Diego Supercomputer Center (SDSC) based at UC San Diego – created a dynamic computer simulation to delineate a key biological process that allows the body to repair damaged DNA.

The simulation describes in detail how a key DNA repair enzyme called thymine DNA glycosylase (TDG) first searches for damaged DNA among a vast background of normal DNA. Once discovered, the enzyme compresses or pinches the DNA so the lesion, a chemically modified or mismatched DNA base, flips out of the DNA base stack and is then pushed into the enzyme’s active site.  Once the aberrant base is captured, the enzyme pulls and snips it away from the rest of the DNA molecule. This initiates subsequent biochemical steps to restore DNA to its original condition, thus completing the repair.

Or, as the paper summarizes: “pinch-push-pull.”

“Our paper describes a novel mechanism for how this lesion search, base interrogation, and flipping occur and explains the origin of the extraordinary specificity of thymine DNA glycolylase,” said  Ivaylo Ivanov, associate professor of chemistry at Georgia State University and the study’s principal investigator. “Deeper understanding of these processes and the respective biochemical pathways could be important medically as well as biologically.”

In essence, TDG has a dual role in the DNA repair process: to maintain the integrity of DNA, and remove epigenetic markers – primarily oxidized derivatives of 5-methyl cytosine – that can silence a gene’s activity without changing its sequence. Put another way, TDG works as a molecular scissor that excises a damaged DNA base or a base carrying an epigenetic modification.

“Understanding the selectivity of TDG is important,” added Ivanov. “These repair proteins are targets for inhibitor development and these can be used as adjuvants in cancer therapy.”

Much of this study builds on ground-breaking discoveries in DNA repair which garnered the 2015 Nobel Prize in Chemistry for Tomas Lindahl, Paul Modrich, and Aziz Sancar for “having mapped and explained how the cell repairs its DNA to safeguard genetic information.”

From a chemical perspective, DNA is a remarkably stable molecule.  But damage to DNA in living cells occurs thousands of times per day, largely stemming from two sources: errors in genome replication or environmental agents such as ultraviolet light, toxic chemicals, ionizing radiation, and the internal presence of reactive molecules.

“Considering how often DNA comes under attack each day, it’s amazing how any lifeform manages to preserve and sustain its genetic code,” said Ivanov.

The genome remains relatively intact over years thanks to a host of repair mechanics circulating in our cells. One of the first and most important processes employed by these repair teams is called base excision repair or BER, identified in 1996 by Lindahl, then director of the Clare Hall Laboratory at Cambridge University. In essence, Lindahl discovered that glycosylases represented the first step in the DNA repair process.

Subsequent research further explained in more detail how the process worked, including computer models based on the X-ray analysis of protein crystals that captured snapshots of the mechanism. However, these static images still failed to describe critical intermediary steps.

In their PNAS article, the Georgia State researchers turned to computational molecular dynamics techniques and SDSC’s Comet supercomputer to simulate the gyrations and seemingly spasmodic movements of TDG as it sculpts or compresses its targeted DNA, flips, and excises detected lesions. Allocations for Comet were provided by the eXtreme Science and Engineering Discovery Environment (XSEDE), funded by the National Science Foundation (NSF).

Among other things, the researchers were able to calculate favorable “free energy profiles” guiding TDG in its lesion search.

“Think of a free energy profile as a sort of map,” said Ivanov. “Valleys on the map are favorable places for the extruded base to reside while it transitions toward the active site of the enzyme. The measure of ‘favorable’ in this case is a thermodynamic quantity we call free energy. The path will follow the lowest free energy regions.”

As summarized by the study: “Our results show that DNA sculpting, dynamic glycosylase interactions, and stabilizing contacts collectively provide a powerful mechanism for the detection and discrimination of modified bases and epigenetic marks in DNA.”

Ivanov said their study would have been difficult to complete if the research team had to rely on local computer resources.

Comet was the best XSEDE resource for us because it provided access to GPUs (graphics processing units) and the code that we used (Amber 16) provides one of the fastest GPU implementations of molecular dynamics” he said.

Also participating in this study, titled “Uncovering universal rules governing selectivity of the archetypal DNA glycosylase TDG”, were Thomas Dodd, Chunli Yan, Bradley Kossman, and Kurt Martin, all from Georgia State University.

Support for the research was provided by a grant from the National Institutes of Health (GM110387) and the NSF (MCB-119521). In addition to allocations from XSEDE, computation resources were provided by the National Energy Research Scientific Computing Center (NERSC), supported by the Department of Energy Office of Science.

As an Organized Research Unit of UC San Diego, SDSC is considered a leader in data-intensive computing and cyberinfrastructure, providing resources, services, and expertise to the national research community, including industry and academia. Cyberinfrastructure refers to an accessible, integrated network of computer-based resources and expertise, focused on accelerating scientific inquiry and discovery. SDSC supports hundreds of multidisciplinary programs spanning a wide variety of domains, from earth sciences and biology to astrophysics, bioinformatics, and health IT. SDSC’s petascale Comet supercomputer is a key resource within the National Science Foundation’s XSEDE (Extreme Science and Engineering Discovery Environment) program.


MATERIALS AND METHODS

Expression and purification of HMO1 protein

Plasmid pJ1870 encoding yeast HMO1 cloned in-frame with an N-terminal His6 tag in expression vector pTEV derived from pET15b (Novagen) was transformed into Escherichia coli BL21(DE3) (Agilent Technologies), and grown in 250-ml LB culture at 37°C with shaking until the culture reached a cell density corresponding to an OD600 of 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and cells were grown at 37°C overnight with shaking, pelleted by centrifugation at 6000 g, and the cell pellet was then resuspended in 10 ml binding buffer (50 mM NaPO4, 300 mM NaCl, pH 7.5) containing 10 mM phenylmethlysulfonylfluoride (PMSF) and passed five times through an Emulsiflex C-5 high-pressure homogenizer (Avestin). The lysate was clarified by centrifugation at 22 000 g for 45 min at 4°C and the supernatant recovered. His6-tagged protein was purified using Ni-NTA agarose resin (Qiagen) per the manufacturer's recommendations. Briefly, washed Ni-NTA agarose resin was added in a 1:4 (v:v) ratio to the lysate, gently rotated at 4°C for 1 h, then loaded onto a 1.5 × 15 cm column. Resin-bound protein was washed with 200 ml wash buffer (50 mM NaPO4, 300 mM NaCl, 20 mM imidazole, pH 7.5). These conditions were sufficient to release bound contaminating DNA. Protein was eluted from the resin with elution buffer (50 mM NaPO4, 300 mM NaCl, 250 mM imidazole, pH 7.5) collecting 2 ml fractions until no protein was detectable with Bradford reagent. Fractions containing the protein of interest were combined and reduced to 2 ml using centrifugal cartridges (Vivaspin), and proteins were dialyzed at 4°C against 1 liter buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT), followed by a second dialysis against the same buffer containing 5% glycerol.

Purified protein was detagged using His6-tagged-TEV protease followed by dialysis into binding buffer to remove residual imidazole. Detagged protein was purified from the His6-tag and His6-tagged-TEV protease by column chromatography as described above, but with elution using nine steps of increasing imidazole concentration between 5 and 250 mM. Fractions with the desired protein were combined and reduced to 2 ml by centrifugal concentration and proteins were dialyzed at 4°C against 1 liter buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 1 mM EDTA and 1 mM DTT, followed by a second dialysis against the same buffer containing 5% glycerol. Protein quality was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, DNA affinity quantitated by electrophoretic gel mobility shifts assays and DNA bending confirmed by enhancement of T4 DNA ligase-mediated DNA cyclization.

Protein–DNA sample preparation for atomic force microscopy

We use 4361 bp linearized plasmid DNA pBR322. Linearization was performed by PvuII digestion followed by phenol extraction ( 20). The freshly cleaved mica surface was exposed to 5 mM Mg 2+ for 20 min at ambient temperature and pressure. The surface was then rinsed with distilled water and air dried. A DNA solution of 0.11 nM was deposited and allowed to incubate for 30 min, then rinsed and dried with argon gas. In order to image the protein-bound DNA complexes, 3 nM protein was incubated with 0.11 nM linearized plasmid pBR322 DNA with 10 mM Tris–HCl (pH 8.0) and 5 mM Mg 2+ , then deposited on the mica surface, left to equilibrate for 20 min, and finally rinsed and dried with argon gas.

Optical tweezers

To investigate and characterize HMO1–DNA interactions, we use dual beam optical tweezers with 830 nm lasers, which can sustain a force up to 300 pN. The experimental setup consists of focusing two laser beams into a 1 μm diameter spot. A bead of high refractive index compared to the surrounding medium will be attracted to the focal point. A second bead is immobilized by a glass micropipette. A high resolution piezoelectric stage (0.15 nm resolution, Npoint) is used to extend the DNA. The force is measured from the refraction of the laser from the polystyrene bead. Once the single molecule is captured between the two beads, the solution around the DNA is exchanged by flowing in 10 times the flow cell volume of protein solution at fixed concentration followed by thermal equilibration and DNA stretching. The buffer solution consisted of 100 mM Na + and 10 mM HEPES pH 7.5. Bridging and looping effects were observed by holding DNA molecules at stretching forces <1 pN.

Here Θ is the DNA fractional site occupancy, KD is the dissociation constant, n is the binding site size (n = 26 bp), c is the concentration and ω is binding cooperativity parameter.

Atomic force microscopy imaging

A Bruker Nanoscope V MultiMode 8 atomic force microscope is used with Peak-Force Tapping™ mode. In this mode, a force curve is obtained at every pixel of the image. The peak force is used as a feedback parameter in order to image topography. The sample can be scanned at lower forces and with shorter contact time, thus protecting delicate samples. For imaging in air, a silicon cantilever is used (resonance frequency = 70 kHz, spring constant = 0.4 N/m and tip radius = 2 nm). The experiments were performed at room temperature. Images are processed using Nanoscope Analysis software, which consists of subtracting the average of each line in order to remove planar artifacts. The scan range used was 1 μm × 1 μm and 2 μm × 2 μm at 512 × 512 pixels and at 1024 × 1024 pixels, respectively. To quantify the atomic force microscopy (AFM) images, DNA molecules were traced and analyzed with NCTracer, software developed by the Neurogeometry Lab at Northeastern University ( 22, 23).


1. Introduction

Double-stranded (ds) DNA can adopt multiple conformations, exhibiting ‘polymorphism’, directly related to the physical properties of the molecule and to its biological function. The most well-known forms of dsDNA are the B-, A- and Z-forms. The B-form is the ‘normal’ DNA found in most biological aqueous contexts. Under reduced water conditions the A-form is favoured, and under certain ionic and base sequence conditions the inverted Z-form prevails. Biological roles of both A- and Z-DNA are possible (Arnott et al. Reference Arnott, Chandrasekaran, Millane and Park 1986 Brown et al. Reference Brown, Lowenhaupt, Wilbert, Hanlon and Rich 2000 Lu et al. Reference Lu, Shakked and Olson 2000 Rich et al. Reference Rich, Nordheim and Wang 1984). Compared with many other polymers, DNA has a spectacular compactness and high stiffness (‘persistence length’) and with a high negative charge density owing to the presence of close phosphate groups in the backbone. The high persistence length is in part due to the electrostatic repulsion of these moieties, partly to the local steric interactions of the coin pile-stacked nucleobases. The great stability of B-form DNA, needed to securely store the genomic information, is due to mainly the stacking of the bases, which in turn is due to hydrophobic effects (requiring surrounding bulk water) and to anisotropic dispersive forces (Friedman & Honig, Reference Friedman and Honig 1995). The base-pair hydrogen bonds, which are important for many biological processes, play only a partial role in the stability of DNA (Guckian et al. Reference Guckian, Krugh and Kool 2000) but are strengthened by the hydrophobic environment in the core of the close-packed bases (see below). DNA can be extended, twisted or unwound during diverse biological processes, such as DNA repair, chromatin compaction and gene regulation. Proteins that interact with DNA are able to exploit the unique structural properties of DNA, although it is not clear to what extent the polymorphic phase map is fully exploited. Taking the particular case of homologous recombination, the recombinase proteins RecA and Rad51 organize themselves in a helical manner around DNA, which is unwound and stretched to about 50% in length compared with the ds B-form (dsDNA). RecA and Rad51 play crucial roles in chaperoning homologous recombination and DNA repair by catalyzing strand exchange in, respectively, prokaryotes and eukaryotes. They use similar molecular reaction mechanisms for the strand exchange reaction and bind first to a single-stranded (ss) part of DNA with high cooperativity, in the presence of ATP, to form a filamentous complex in which DNA adopts an extended conformation (Flory et al. Reference Flory, Tsang and Muniyappa 1984). The ssDNA–RecA filament then interacts with an incoming dsDNA to form an ssDNA–RecA–dsDNA complex (Kiianitsa & Stasiak, Reference Kiianitsa and Stasiak 1997) and, if the two DNAs have the same or similar sequence, strand exchange occurs.

Twenty-five years ago, we studied shear-aligned fibres of Escherischia coli RecA complexes with both ss as well as dsDNA in aqueous solution by small-angle neutron scattering (SANS) and UV linear dichroism (LD) spectroscopy under identical flow conditions (Nordén et al. Reference Nordén, Elvingson, Kubista, Sjöberg, Ryberg, Ryberg, Mortensen and Takahashi 1992). The orientation created a well-resolved SANS pattern, where the helical diffraction cross provided exact information about the helix pitch and, most importantly, yielded the flow orientation distribution, making it possible to translate the LD signals into average base-plane orientations (Hagmar et al. Reference Hagmar, Norden, Baty, Chartier, Takahashi, Nordén, Baty, Chartier and Takahashi 1992 Nordén et al. Reference Nordén, Elvingson, Kubista, Sjöberg, Ryberg, Ryberg, Mortensen and Takahashi 1992). Electron microscopy had revealed that the DNA was extended by approximately a factor of 1·5 (Stasiak & Di Capua, Reference Stasiak and Di Capua 1982 Stasiak et al. Reference Stasiak, Di Capua and Koller 1981), but surprisingly the base orientation concluded from LD did not show any inclination of the base planes (neither for ss- nor for dsDNA) as would be expected for a continuously stretched and unwound DNA form (Hagmar et al. Reference Hagmar, Norden, Baty, Chartier, Takahashi, Nordén, Baty, Chartier and Takahashi 1992 Nordén et al. Reference Nordén, Elvingson, Kubista, Sjöberg, Ryberg, Ryberg, Mortensen and Takahashi 1992, Reference Nordén, Wittung-Stafshede, Ellouze, Kim, Mortensen and Takahashi 1998). This observation remained puzzling until 2002, when the application of systematically mutated aromatic residues in RecA allowed a three-dimensional model to be constructed for the aqueous solution structures of RecA-dsDNA and RecA-ssDNA (Morimatsu et al. Reference Morimatsu, Takahashi and Nordén 2002). From LD results combined with crystal data for RecA (Story et al. Reference Story, Weber and Steitz 1992), a model emerged in which the DNA was accommodated in an ordered way inside a helical arrangement of RecA monomers allowing the bases to be perpendicular (Morimatsu et al. Reference Morimatsu, Takahashi and Nordén 2002). A later crystal structure of RecA–dsDNA and RecA–ssDNA confirmed our conclusion: a near perpendicular nucleobase orientation and clustering of triplets of bases stacked approximately as in B-form DNA (Chen et al. Reference Chen, Yang and Pavletich 2008) (Fig. 1). Using LD spectroscopy together with site-specific tyrosine mutations, we found a similar nucleobase organization, perpendicular to the fibre axis of the complex, for the dsDNA complex with Rad51 in solution (Reymer et al. Reference Reymer, Frykholm, Morimatsu, Takahashi and Nordén 2009). For the Rad51 complex with ssDNA, however, a perpendicular base orientation was observed only in the presence of activating factors, such as Ca 2+ ion or Swi5/Sfr1 protein (Fornander et al. Reference Fornander, Renodon-Corniére, Kuwabara, Ito, Tsutsui, Shimizu, Iwasaki, Nordén and Takahashi 2014). Recent cryo-EM high-resolution structural analyses of activated human Rad51 in complex with DNA have demonstrated conserved features with the RecA system (Xu et al. Reference Xu, Zhao, Xu, Zhao, Sung and Wang 2017).

Fig. 1. The DNA structure inside a RecA-dsDNA filament according to crystal structure (left) (Chen et al. Reference Chen, Yang and Pavletich 2008) compared with free-solution double-stranded B-form DNA (right).

In 2012, we identified using single-molecule force spectroscopy on short synthetic DNAs, in the absence of recombination proteins, the existence of an overstretched state of DNA, which we shall here return to and study in detail. All evidence indicates it is a thermodynamically well-defined conformation. We will call it Σ-DNA, and it consists of a ca 50% extended stable conformation of ds (base-paired) GC-rich DNA, at a transition force of ca 64 pN applied to the 3′−3′ strands (Bosaeus et al. Reference Bosaeus, El-Sagheer, Brown, Smith, Akerman, Bustamante and Norden 2012). The Σ-form may be considered a special case of the wider group of stretched DNA forms that have been called ‘S-DNA’ some of which, though, appear less well defined. In contrast to the Σ-form observed for base-paired GC-rich DNA stretched along the 3′–3′ ends, S-DNA is usually observed as a 70% elongated form when stretching a long plasmid or phage DNA (Cluzel et al. Reference Cluzel, Lebrun, Heller, Lavery, Viovy, Chatenay and Caron 1996 Williams et al. Reference Williams, Rouzina and Bloomfield 2002) and, at least in AT-rich DNA, S-DNA appears to involve denaturation (Bosaeus et al. Reference Bosaeus, El-Sagheer, Brown, Smith, Akerman, Bustamante and Norden 2012, Reference Bosaeus, El-Sagheer, Brown, Åkerman and Nordén 2014). The fact that the Σ-form has so long escaped discovery is thought to be due to that it is first recently stretch experiments on short synthetic DNA have been possible with high accuracy (Bosaeus et al. Reference Bosaeus, El-Sagheer, Brown, Smith, Akerman, Bustamante and Norden 2012, Reference Bosaeus, El-Sagheer, Brown, Åkerman and Nordén 2014).

The degree of extension that we observe for Σ-DNA, compared with B-DNA, is within experimental error the same as that found in complexes with the recombinase enzymes RecA and Rad51. One may thus ask if this is just a coincidence or if this structural distortion is somehow related to biological function. Here we further analyze previous data and perform some new strategic single-molecule and computational experiments in order to assess the possibility that Σ-DNA may have fundamental roles in biology.

Our hypothesis is that Σ-DNA is an inhomogeneous structure consisting of stacked triplets of nucleobases, with base planes arranged perpendicularly as in B-DNA, and that these triplets are separated by empty gaps. Such a triplet structure may have a biological role in enhancing the recognition of complementary base sequence and promote the strand exchange process in gene recombination. We further speculate that the base triplets separated by gaps may be a physical origin of the occurrence of three letters in the genetic code.


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Regulation of Gene Transcription

Virtually every adaptation and developmental process originates at the level of gene regulation by transduction of extra- or intracellular signals. Transcription is the major target of regulation of gene expression. Our research interest covers modulation of transcription by DNA control elements and regulatory proteins, for example, repressors, activators, terminators, and antiterminators and their cognate signal molecules. We have demonstrated transcriptional regulation both at the level of initiation by activators and repressors and at the level of elongation by terminators and antiterminators in the gal operon which encodes enzymes of D-galactose metabolism in Escherichia coli. Presumably because of the amphibiotic nature of the biochemical pathway in carbon metabolism, the operon shows a multitude of controls at all levels of transcription and has been a paradigm system for studying control of transcription. The operon is transcribed from two promoters which are subject to both negative and positive control by at least four proteins, Gal repressor, Gal isorepressor, cyclic AMP receptor protein, and bacterial histone like protein, HU.

Major Findings:

  • The two promoters of the gal operon are regulated by formation of a DNA loop. We have determined the structure, assembly and dynamics of the DNA loop, which constitute a higher order nucleoprotein complex (Gal repressosome). The characterization involves genetic and biochemical analysis, evaluation of DNA elastic energy, atomic force microscopy and in vitro single molecule analysis. In the Gal repressosome a DNA loop is formed by the interaction of two GalR dimers, bound to two spatially separated operators, OE and OI, flanking the gal promoters. Structure-based genetic analysis have indicated that GalR homodimers interact directly and form a V-shaped stacked tetramer in repressosome, which is further stabilized by HU binding to an architecturally critical position on the DNA. In this scheme of GalR tetramerization, the alignment of the operators in the DNA loop could be in either parallel (PL) or antiparallel (AL) mode. As each mode can have two alternative geometries differing in the mutual stacking of the OE - and OI -bound GalR dimers, it is feasible to have four different DNA trajectories in the repressosome. Our results show that OE and OI adopt a mutual AP orientation in an under-twisted DNA loop, consistent with the energetically optimal structural model. In this structure the center of the HU-binding site is located at the apex of the DNA loop. The approach reported here can be used to distinguish between otherwise indistinguishable DNA trajectories in complex nucleoprotein machines.
  • We have demonstrated that a regulator inhibits or stimulates transcription initiation by disabling or stimulating RNA polymerase activity at a post-binding step by directly or indirectly altering the specific domain of RNA polymerase by a direct GalR-RNA polymerase contact(s) to an unfavorable or to a more favorable state, respectively. Base unpairing during isomerization of the closed to open complex at a promoter is an asynchronous process with a rate-limiting step as observed by 2, AP fluorescence kinetics.
  • We have shown that a stretch of purines present in a specific transcribing region can override transcriptional pause signals by preventing RNA polymerase back-tracking created by the a pause signal.
  • We have determined the axiom that determines the start point of transcription by RNA polymerase.
  • Small molecules generally activate or inhibit gene transcription as externally added substrates or as internally accumulated end-products, respectively, Rarely has a connection been made that links an intracellular intermediary metabolite as a signal of gene expression. We have shown that a perturbation in the critical step of a metabolic pathway changes the dynamics of the pathways leading to accumulation of an intermediary metabolite. This accumulation causes cell stress and transduces signals that alter gene expression so as to cope with the stress by restoring balance in the metabolite pool. This underscores the importance of studying the global effects of alterations in the level of intermediary metabolites in causing stress and coping with it by transducing signals to genes to reach a stable state of equilibrium (homeostasis).
  • From modeling studies, we have concluded that the structure of the regulatory network is insufficient for the determination of signal integration. It is the actual structure of the promoter and regulatory region, the mechanism of transcription regulation and the interplay between the transcription factors that shape the input function to be suitable for adaptation.

Regulatory Biology of Bacteriophage λ
The lysis-lysogeny decision of bacteriophage λ is a paradigm for developmental genetic networks. Two key features characterize the λ network. First, after infection of the host bacterium, a decision between lytic or lysogenic development is made that is dependent upon environmental signals and the number of infecting phages per cell. Second, the lysogenic prophage state is very stable. The CI and Cro regulators define the lysogenic and lytic states, respectively, as a bistable genetic switch. Whereas CI maintains a stable lysogenic state, recent studies indicate that Cro sets the lytic course not by directly blocking CI expression but indirectly by lowering levels of CII, which activates CI transcription. We are investigating how a relatively simple phage like λ employs a complex genetic network in decision-making processes, providing a challenge for theoretical modeling. Recently, we have made the following observations:

  • The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. Furthermore, labeling of λ DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to λ developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.
  • The lysogenic state of bacteriophage λ is exceptionally stable yet the prophage is readily induced in response to DNA damage. This delicate epigenetic switch is believed to be regulated by two proteins the lysogenic maintenance promoting protein CI and the early lytic protein Cro. First, we have confirmed the previous observation that a DNA loop mediated by oligomerization of CI bound to two distinct operator regions OL and OR, increases repression of the early lytic promoters and is important for stable maintenance of lysogeny. Second, we have shown that the presence of the Cro gene might be unimportant for the lysogenic to lytic switch during induction of the lambda prophage.
  • The spontaneous derepression of a λ prophage is less than mutational frequency. We have found by in vitro transcription system, that the extreme stability is because of DNA loop formation by interaction of CI-dimers to the right and left λ operators in the prophage state in a cross-wise cooperativity. Multilevel co-operativity in DNA loop discourages CI dissociation from the operators in stabilizing the prophage. It also shields prophage repression against lowering CI concentration and operator mutations.
  • We have studied the stability of a DNA loop by tethered particle motion experiments in single DNA molecule experiments to study the dynamics of λ DNA looping. Modeling the thermodynamic data thus obtained established that the loop that involves 12-CI monomer is the stable one. The one that involves only 8 monomers has a positive free energy.

Bacterial Nucleoid
Bacterial necleoid organization is believed to have minimal influence on the global transcription program. Using an altered bacterial histone-like protein, HUα, we show that reorganization of the nucleoid configuration can dynamically modulate the cellular transcription pattern. The mutant protein transformed the loosely packed nucleoid into a densely condensed structure. The nucleoid compaction, coupled with increased global DNA supercoiling, generated radical changes in the morphology, physiology, and metabolism of wild-type Escherichia coli K-12. Many constitutive housekeeping genes involved in nutrient utilization were repressed, whereas many quiescent gene associated with virulence were activated in the mutant. We propose that, as in eukaryotes, the nucleoid architecture dictates the global transcription profile and, consequently, the behavior pattern in bacteria. We are investigating whether the E. coli nucleoid has a defined structure. In support of that we have made the following observations:

  • Molecular mechanisms of bacterial chromosome packaging are still unclear. Among the factor facilitating DNA condensation may be a propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity. We analyzed positioning of A-tracts (the sequence motifs introducing the most pronounced DNA curvature) in the Escherichia coli genome. A-tracts are over-represented and distributed 'quasi-regularly' throughout the genome, including both the coding and intergenic sequences. There is a 10-12 bp periodicity in the A-tract positioning indicating that the A-tracts are phased with respect to the DNA helical repeat. The latter feature was revealed with the help of a novel approach based on the Fourier series expansion of the A-tract distance autocorrelation function. Since the A-tracts introduce local bends of the DNA duplex and may serve as binding sites for the nucleoid-associated proteins that have affinities for curved DNA. We have suggested that long clusters of the phased A-tracts constitute the 'structural code' for DNA compaction by providing the long-range intrinsic curvature and increasing stability of the DNA complexes with architectural proteins.
  • We have determined the crystal structure of the Escherichia coli nucleoid-associated HU protein by x-ray diffraction and observed that the heterodimers form multimers with octameric units. It is of special importance that one of the structures forms spiral filaments with left-handed rotations. A negatively superhelical DNA can be modeled to wrap around this left-handed HU multimer, thus providing the structural explanation for the classical property of HU to restrain negative supercoils in DNA.
  • The contribution of global nucleoid organization in determining cellular transcription programs is unclear. Using a mutant form of the abundant nucleoid-associated HU we showed that nucleoid remodeling by the mutant protein re-organizes the global transcription pattern. We have demonstrated that, unlike the dimeric wild-type HU, the mutant HU is an octamer and wraps DNA around its surface. The formation of wrapped nucleoprotein complexes by the mutant HU leads to a high degree of DNA condensation. The DNA wrapping in the mutant is right-handed, which restrains positive supercoils.

Bacteriophage Applications
Bacteriophage (phage) have been used in treatment of bacterial infections in humans since their discovery many decades ago, but the practice was discontinued because of lack of scrutiny required for Western medicine, and because of the discovery of potent antibiotics. Widespread occurrence today of drug resistant pathogens motivated us to attempt to revive phage-based diagnosis and treatment of bacterial infections using E. coli and Yersinia pestis as model systems.

In these studies, we used λ and T7-like phages that we mutated or engineered for use in therapy of experimental bacteremic animals, in the investigation of mutation rates in cancer cells and in the detection of E. coli and Yersinia pestis in clinical and environmental samples.

  • Analysis of protein-protein interactions is critical in proteomics and drug discovery. Usage of a bacterial or yeast 2-hybrid system to detect protein-protein interaction is limited to an in vivo environment. Using the λ- display system, we have developed a phage-based 2-hybrid system for in vitro investigations.

Collaborators on this research include Victor Zhurkin, CCR, NCI, NIH, and Laura Finzi, Emory University, Atlanta, GA.


DISCUSSION

The multi-step process of change of linking number catalyzed by topoisomerases is likely to require participation of different functional groups in different steps of the reaction. In the current mechanism of E.coli topoisomerase I, Tyr319 is responsible for nucleophilic attack on DNA and formation of the covalent bond between the enzyme and the 5′ phosphate of the cleaved DNA (7). Glu9 found also in the active site has been implicated in the process of DNA cleavage (9,10). In its proximity, Ser10 and Lys13 are conserved residues with side chains that could also participate in the catalytic process. Results presented here on the site-directed mutants support a role for these two residues in the DNA cleavage step of the enzyme mechanism. Altering these residues to alanines did not affect non-covalent binding to DNA, but significantly reduced the formation of the cleaved DNA complex. The changes of Ser10 to Thr and Lys13 to Arg had more severe effects on the enzyme, reducing also the non-covalent binding of enzyme to DNA. Although proteolytic digestion of the S10T and K13R mutants did not indicate severe defects in protein folding, the changes in the side chains in these substitutions could potentially affect the structure of the junction region between domains I and III at the active site, affecting also the non-covalent interaction of the enzyme with the single-strand of DNA that has to be positioned at the active site for cleavage.

In the crystal structure of E.coli topoisomerase III complexed with ssDNA (17), the lysine residue (Lys8) that corresponds to Lys13 in topoisomerase I is in a position to interact with the phosphate group of the scissile bond ( Figure 1 ). The importance of this residue in DNA cleavage as suggested by the structural data (17) is now demonstrated biochemically in this study.

In the structure of the 67 kDa N-terminal fragment of topoisomerase I, the side chains of both Ser10 and Lys13 are in the vicinity of Tyr319 with their relative positions switched when compared with Lys8 and Ser10 of topoisomerase III ( Figure 1 ). The positively charged side chain of Lys13 is expected to interact with either the scissile phosphate and/or the acidic pocket of Glu9/Asp111/Asp113 via electrostatic interactions. The results from our molecular modeling simulations support the interaction of Lys13 with the acidic pocket, particularly with Glu9 and Asp111. Although our models show that the distance from Lys13's amide group to the scissile phosphate is 𢏇 Å, conformational changes in the enzyme might easily halve the distance between these groups. Modeling studies currently underway simulating the transition of the ‘open’ complex to the 𠆌losed’ complex may reveal further details of these proposed interactions.

In the crystal structure of the topoisomerase III𠄽NA complex (17), the side chain of Ser10 does not appear to be in a position to participate directly in DNA cleavage. Nevertheless, this residue is in proximity to the phosphate immediately 3′ of the scissile phosphate and a water-mediated hydrogen bond is observed between Ser10 and this phosphate. The topoisomerase enzyme used in the crystallization of this complex has phenylalanine substituted for the active site tyrosine. It is also in the absence of the Mg 2+ ions required for catalytic activity and using a short oligonucleotide substrate. It cannot be ruled out that with the conformational changes expected from topoisomerase in the presence of either an active site tyrosine, Mg 2+ ions, or with longer ssDNA substrates that the position of the side chain of this serine residue could change to participate more directly in DNA cleavage. In our simulated topoisomerase I complex with a longer oligonucleotide, the side chain of Ser10 forms a hydrogen bond directly with the phosphate 3′-adjacent to the scissile phosphate. This interaction appears to be crucial to the positioning of the DNA substrate in the active site when this hydroxyl was removed in the S10A mutant, deoxyribose 6 located 5′ to the scissile phosphate shifted from a ‘northern’ pucker to the ‘southern’ pucker typical of B-form DNA. In the F328Y topo-III and H365R topo-I cocrystals (17,18), the analogous deoxyriboses are also observed in a ‘northern’ pucker conformation. The ‘northern’ ribose conformations uniquely position O3′ along the equitorial plane of the ribose which orients the adjacent phosphate oxygens towards the tyrosine nucleophile. Thus, Ser-10 aides the positioning of the scissile phosphate for DNA cleavage and may account for the conserved presence of this serine residue among type IA topoisomerase sequences. We propose that helix O and Ser10, respectively are the 5′ and 3′ ends of a clamp that holds the DNA substrate in the active site properly positioned for cleavage.

Topoisomerase mechanisms share a common feature with some enzymatic phosphoryl transfer reactions in the formation of a covalent intermediate. In these two-stage mechanisms, an enzyme nucleophile first attacks a phosphorus to form a stable covalent intermediate. In the second stage of the reaction, the covalent intermediate is in turn attacked by a nucleophile to generate the free enzyme again and reaction products (32). Enzymatic phosphoryl or nucleotidyl transfer reactions are often facilitated by surrounding the attacked phosphate with positively charged groups including the side chain of lysine residues (33,34). In the study of the mechanism of enzymatic phosphoryl transfer by phosphoserine phosphatase, a hydrogen bond between an active site serine residue and a non-bridging oxygen of the phosphoryl group, as well as salt bridge interactions by an active site lysine residue, have been proposed to be important for ground-state stabilization (32,35). Therefore examples of utilization of a combination of conserved active site serine and lysine residues to catalyze the formation of a phosphoryl-enzyme covalent intermediate are already known.

A role for the conserved Arg321 with its positively charged guanidinium group close to the active site tyrosine hydroxyl in promoting nucleophilic attack by Tyr319 by stabilization of the negatively charged phenolate ion has been proposed for the mechanism of E.coli DNA topoisomerase I (9,13,24). The corresponding arginine Arg330 in E.coli DNA topoisomerase III was observed to contact the scissile phosphate in the complex with ssDNA (17). A similarly positioned arginine residue may have similar functions in type II topoisomerases (36,37). Based on the structure of E.coli DNA topoisomerase III complexed to ssDNA, it was also proposed that Glu7 of topoisomerase III (corresponding to Glu9 of E.coli DNA topoisomerase I) acts as a general acid catalyst by donating a proton to the leaving 3′-oxygen atom of the scissile bond during DNA cleavage and may also act to abstract a proton from the free 3′-hydroxyl group during nucleophilic activation for DNA religation (17). Mutagenesis studies of His365 of E.coli DNA topoisomerase I led to the proposal that His365 donates a proton to Asp111 which then relays the charge to Glu9 for its protonation (14). The stable salt bridge that Lys13 forms with Glu9 and the observed activity defects in alanine mutagenesis suggests that Lys13 may participate in the proton transfer.

This study provides biochemical evidence that contact with the 3′-downstream phosphate involving Ser10 may also be important for the cleavage step of catalysis and that a second positively charged side chain may be used in the active site to donate a proton and/or contact the scissile phosphate. Similar roles have also been suggested for other active site residues (9,10,18). Active site residues in the topoisomerase may play multiple roles, further underscoring the finding that few alanine mutations completely eliminate relaxation activity (9).

Additional structural information on the different conformations of the enzyme and its complex with DNA, molecular modeling of the different topoisomerase states and the proposed catalytic mechanisms, and mutagenesis data on other conserved amino acids with potential catalytic roles will help elucidate the mechanism of this class of enzyme.


Watch the video: What happens when your DNA is damaged? - Monica Menesini (May 2022).


Comments:

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  4. Flannagan

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  6. Sifiye

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  7. Walter

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