Chromatin Chapter 1: DNA Organization, the Nucleosome, and Histones

Chromatin is one of those things that I never really paid any attention until I suddenly realized how important and interesting it is.  How is it that so much information (DNA) can be stored in such a small space (the nucleus)?  How is it that this information is used when it is so tightly packaged?  It may sound nerdy, but I’m still in awe at how important chromatin really is.  This next set of posts will explore the basics of chromatin and begin to touch on the effects it has on genes and cells.  I’ve already mentioned how it affects DNA repair, but the process has even more far-reaching effects.

DNA Organization

Chemically, DNA and RNA are composed of sugar phosphate backbones with nitrogenous bases attached.  The sugar comes in the form of ribose (in the case of RNA) or deoxyribose (in the case of DNA).  Deoxyribose lacks the 2’ hydroxyl group on ribose.  The ribose / deoxyribose sugars are connected via a phosphate linkage (PO₄) via the 3’ and 5’ hydroxyl groups.  The 1’ hydroxyl group is where the nitrogenous base attached.  Purines contain a purine ring and come in the form of adenine and guanine.  Pyrimidines consist of cytosine, uracil, and thymine.  The ribose / deoxyribose connected to the base and the phosphate are considered nucleic acids.

Nearly everyone knows the famous story of Watson and Crick and their discovery of the structure of DNA.  They hypothesized (correctly so) that DNA consists of a double-helix held together by the hydrogen bonds formed by the nitrogenous bases (adenine to thymine; guanine to cytosine).  This double helix is antiparrallel, right-handed, and has polarity: the 5’ end is attached to a phosphate group, while the 3’ end consists of a free hydroxyl group.  The helix turns once every 10.5 bases at a total distance of 36 Å, with a 3.4-Å rise per base and a width of 20 Å.  The entire helix is negatively charged due to the phosphate groups that connect the sugars. 

DNA does not exist in the cell as a free-floating molecule.  Instead, it is shaped and organized by chromatin, the makeup of the chromosomes consisting of the DNA itself and the attached proteins.  In the case of humans, unraveled DNA measures about two meters in length, but cell nuclei are, at most, 10 μm.  Therefore, the cell must attain a 10,000-fold compaction while still performing all the requirements for the cell.  To accomplish this, the cell uses the chromatin hierarchy, composed of five orders of organization.

The Nucleosome

The first order of chromatin packaging is the nucleosome, the most basic organization mechanism used to compact the DNA.  The nucleosome packages 147 bp of DNA wrapped on “beads” of eight histone proteins (making an octamer). These octamers are positioned at intervals on the DNA, and,  if the DNA is spread, the  nucleosomes attached to the DNA look like beads on a string.  The nucleosome consists of histones H2A, H2B, H3, and H4, and nucleosomes are attached to each other by linker histone H1.  Linker and nucleosomal histones are made throughout S phase, when new DNA is synthesized and must be compacted.   Histones can be modified in several ways to affect the structure and dynamics of the DNA.  The proteins have largely been conserved through evolutionary history but variants do exist.  These variants are synthesized mostly during interphase and insert into mature chromatin via chromatin remodeling complexes.  One of these variants, H2A.Z,  limits chromatin condensation; another variant H2A.X is involved in DSB response.  H3.3 can be found in long-term active chromatin.  In general, these variants are involved in changes in chromatin that remain for long periods of time in the cell. 

In addition to histones, a number of other proteins bind DNA and are included in the chromatin.  Namely, the high mobility group proteins (HMGs), polymerases, and DNA repair enzymes interact with the DNA and the chromatin. 

The DNA itself is wrapped around the nucleosome 1.75 times, with about 60 bp of DNA between nucleosomes and associated with linker histone H1.  The nucleosomes contain groves that fit the DNA between H2B and H4 and H4 and H3.  The octamer itself exists as two H2A/H2B dimers and one H4-H3-H3-H4 tetramer.  

Thermodynamics Take II: The Second Law, Gibbs-Helmholtz Equation, State Functions

This will be the final post about thermodynamics, but related posts (namely on some biophysics-type topics) will be posted down the line.  The equations presented are summarized at the bottom of this post.

How do biological systems follow this second law?  After all, we’re all (rather) organized beings, and there had to be a decrease in entropy when our DNA, lipids, proteins, and all the other biomolecules organized in our bodies.  However, biological systems follow the law because they are open systems and take in (exchange) energy from the environment.  The entropy of the surroundings increases even though the entropy of the system (such as the human body) decreases. 

As mentioned, entropy is a measure of disorder, in a way.  Entropy can be calculated as

S = k_B ln W

where k_B = 1.38x10^-23J/K (Boltzman’s constant) and W is the number of ways to arrange a state.  If you wanted to calculate this, you could, but we are more concerned with changes in entropy than the actual entropy inherent in a molecule.  For example, in the case of glucose (C₆H₁₂O₆) and six oxygen molecules being converted to six molecules of CO₂ and H₂O, the entropy will increase because there are 12 molecules of carbon dioxide and water, but there are only 7 of glucose and oxygen. 

Relationship of Free Energy, Entropy, and Enthalpy

All of the above thermodynamic properties are related in what is called the Gibbs-Helmholtz equation, which states:

ΔG = ΔH – T ΔS

where T is the temperature in Kelvin (always a positive value).  Considering this equation further, one can see that ΔG is negative (a process is spontaneous) if ΔH is negative and ΔS is positive.  On the other hand, if ΔH is positive and ΔS is negative, ΔG is positive and the process is not spontaneous (it would require energy input for it to occur).  In fact, if ΔG is positive, the reverse process is spontaneous (conversion of products to reactants).  If ΔH and ΔS have the same sign, ΔG could be either positive or negative, depending on the magnitude of the values. 

Given the Gibbs-Helmholtz equation, one can quickly calculate the transition temperature at which point a reaction (or process) changes from spontaneous to non-spontaneous as:

T = ΔH / ΔS

which occurs when ΔG = 0. 

Reactions that are considered entropy driven are those that have a positive ΔS and ΔH values and a negative ΔG value, indicating that the reaction is spontaneous and that the change in entropy is the major factor contributing to the spontaneity.  In contrast, an enthalpy-driven reaction is one in which ΔH is negative and ΔS is positive, meaning that ΔG is negative.  In this case, the negative free energy value is due solely to the negative value of the change in enthalpy.

All of the above terms (enthalpy, entropy, free energy) are considered state functions, meaning that the values of enthalpy, entropy, and free energy depend on the system’s current state, not the path to get to that state. Due to this convenient rule, we can calculate the free energy of formation (ΔG_f^o) for various compounds by adding and subtracting free energies of the component molecules at the biochemical standard state (1 atm, 25^oC, pH 7.0). 

One important result of free energy being a state function is that free energy changes are additive: chemical reactions can be “added” (add reactants to reactants, products to products) and the total free energy change is the sum of the component reactions.  

Summary of equations:

ΔG = ΣG_products - ΣG_reactants

ΔH = ΣH_products - ΣH_reactants

ΔS = ΣS_products - ΣS_reactants

ΔS_universe = ΔS_system + ΔS_surroundings > 0

S = k_B ln W

Gibbs Helmholtz Equation: ΔG = ΔH – T ΔS

Transition Temperature: T = ΔH / ΔS

Thermodynamics Take I: The Basics, Free Energy, Entropy, Enthalpy

Today’s post will be a slight departure from cell biology and genetics and will focus, instead, of some of the basics of biochemistry.  It is true that I disliked biochemistry, and that’s putting it lightly, but it’s still something that is important (how important is another question) to understand.  I took a few days off from updating but I return with more review fun.  This set of posts will include a number of equations that I will summarize after all of the thermodynamics notes have been posted.  Also, the illustrations for these posts will be simplistic, but if you have some better ideas of how to illustrate thermodynamics, I'd like to know because I'm coming up with nothing...

Thermodynamics is the study of the relationships between energy and chemical processes.  This energy can come in two distinct forms, namely potential and kinetic energy.  Most of use probably learned in high school physics class that potential energy is stored energy, as in a ball that you hold above the ground  has the potential to fall to the ground and therefore has stored / potential energy.  Kinetic energy is the energy of motion, which you probably learned as the energy of a moving ball during that same physics lesson.  In terms of biology, however, potential energy has deeper meaning (it’s more than balls), such as stored energy in chemical bonds (ATP), concentration gradients, and electrical potential via ion gradients.  Kinetic energy in terms of biology can come in the form of heat energy due to atomic motion (just as we learned in physics) or in radiant energy, including electromagnetic radiation (light).

The First Law of Thermodynamics: energy is conserved

Sure, there are about a billion ways of rephrasing the first law of thermodynamics, but, put simply, energy is conserved.  In terms of chemical reactions, there is what is called the free energy (Gibbs free energy), or G.  Gibbs free energy is the work that is available to do work.  In a reaction or process, the change in free energy is calculated as:

ΔG = ΣG_products - ΣG_reactants

When ΔG is negative (ΔG < 0), the reaction or physical process is spontaneous, though that is not to say that it will happen quickly.  A negative ΔG value simply means that energy need not be added to the system for it to react.  A negative ΔG value is considered exergonic, while a positive ΔG value is endergonic.  A positive value for ΔG means that energy must be added to the system for the process.  At equilibrium in a reaction, ΔG is zero, meaning that neither the amount of products or reactants is changing: no energy is consumed or released.

Free energy, G, can be further broken down into enthalpic, H, and entropic, S, components. 

Enthalpy is a measure of the internal energy of a system in kcal/mol (or kJ/mol).  At constant temperature and pressure, enthalpy is equivalent to the heat absorbed or released and

 ΔH = ΣH_products - ΣH_reactants

Endothermic reactions are those that absorb heat (ΔH > 0); exothermic reactions release heat energy (ΔH < 0).  Adding heat will affect the equilibrium of the reaction (whether it favors products or reactants).  Because endothermic reactions require energy for the reaction to occur, raising the temperature favors the reactants forming products.  Conversely, adding heat to an exothermic reaction will favor the formation of reactants from products because, in fact, heat is a product of the reaction.

Entropy is a measure of the randomness of a system.  While many would dispute this definition, for our purposes it works (and this write-up is not about semantics).  Entropy is measured in cal/mol K (J/mol K).  As you would expect,

ΔS = ΣS_products - ΣS_reactants

The second law of thermodynamics states that entropy of a system and its surroundings always increases in a reaction.  The disorder will tend to a maximum and ΔS > 0.  Therefore, if we want a more ordered system (such as in the polymerization of DNA from dNTPs), we have to add energy.  This may seem counterintuitive to some degree, but one must remember that we are considering the entropy of both the system and its surroundings:

ΔS_universe = ΔS_system + ΔS_surroundings > 0

Cell Cycle III: Entering and Exiting Mitosis (and the Events Between)

Today is the final part of the cell cycle that I will be writing, though more information about cell division will be posted eventually in the meiosis study notes.  I find that the pathways involved in mitosis are rather easy to understand, as everything fits together - it just takes some effort to realize how everything works with everything else. 

When the cell is ready to begin anaphase, the sister chromatids must be aligned at the metaphase plate, and the proper Cdk/cyclin must remain activated (Cdc2/cyclin B).  When anaphase is to begin, APC/C (anaphase promoting complex / cyclosome) activates via phosphorylation from Cdk/cyclin and association with a cofactor.  APC/C acts as a ubiquitin ligase and is involved in poly-ubiquitinating proteins to target them for degradation.  When phosphorylated APC/C binds Cdc20, it targets securin for degradation.  Securin typically holds a protein called separase.  When securin releases separase, the protein cleaves cohesion complexes and separates sister chromatids.  This prompts the cell to enter anaphase when the sister chromatids migrate away from each other.  This process is regulated by Bub and Mad2, which act to inhibit the action of APC/C^Cdc20.  When a checkpoint is activated by a sister chromatid not being bound to a kinetochore MT, for example, it actively signals to Bub, which activates Mad2 to activate Mad2*.  Mad2* is involved in inhibiting the APC/C^Cdc20 complex.  The end result is that the sister chromatids do not separate. 

During anaphase, the microtubules are undergoing a number of changes.  Initially in anaphase (Anaphase A), the kinetochore microtubules, bound to the kinetochores of the sister chromatids, begin to shorten, pulling the chromatids apart.  During anaphase B, the astral microtubules remain in place, maintaining the spindle pole in place.  The polar microtubules begin to push on each other by elongating, which pushes the spindle poles apart and promotes separation of the nuclei.  When the spindles have moved apart and the chromatids have separated, APC/C targets Ase1p for degradation, which leads to breakdown of the spindle. 

When cohesion is cleaved by separase (see above), Cdc14 phosphatase is released from the nucleolus.  This event is the signal to end mitosis by inducing expression expression of Sic1, which acts to deactivate MPF.  Cdc14 dephosphorylates and activates Cdh1, which complexes with APC/C to target MPF for degradation.

With MPF degraded proteolytically, the nuclear envelope is free to reform.  Constitutive phosphatases dephosphorylate the lamins and nuclear pore complexes.  This results in the formation of karyomeres, which are small vesicles (with nuclear pores in them) that form around the sister chromatids.  The karyomeres then fuse to reform the nuclear envelope. 

In addition to the nuclear lamins and pore complexes, myosin light chain is also dephosphorylated when MPF is degraded.  This results in the activation of the protein and the initiation of cytokinesis when the contractile ring begins to form.

A quick summary…

Phase of the Cell Cycle	Description
Interphase	The cell is preparing to duplicate; centrosomes appear outside the nucleus
Early Prophase	Spindle poles form and sister chromatids condense
Late Prophase	Sister chromatids are condensed and begin to be attached by kinetochore MTs
Metaphase	Sister chromatids align at the metaphase plate
Anaphase	The cell begins to divide the sister chromatids by releasing them to each pole
Telophase	The nuclear envelope reforms and the cells begin to structurally split

The cell cycle isn’t so scary, but it certainly is complex, and not all of the components and regulatory mechanisms have been discussed here (or discovered in the literature either!).  Nonetheless, it is crucial to understand the basics of the cell cycle because it is so intimately related to nearly all biological processes.

Cell Cycle II: Condensins, Cohesins, Microtubules, and All the Fun Stuff

I'm going to try to make this blog post a little different with a short introduction before jumping right into my writings and ramblings about the cell cycle.  This post is the second of three concerning the cell cycle, though more information will be eventually posted that is somewhat related to this topic (notably the meiosis notes).  I haven't drawn a diagram for this particular post, but there are not many mechanisms involved.  Tomorrow's post will include a cool drawing of separase, securin, and a bunch of other cool stuff.  

When I was in college and learning the cell cycle, my professors weren't interesting or very organized.  Therefore, I've tried to organize this in a way that makes sense to me.  Since this organization might not make sense to everyone else, I'll be including a list of links to review articles and other websites that concern the cell cycle when all of the sections have been posted.

Now that we’ve covered the “motor” of the cell cycle, we need to consider regulation in terms of different stages of the cell cycle and the associated checkpoints.  These checkpoints influence the activation of Cdk/cyclin and affect whether the cell arrests or progresses through the cycle.  During S phase, the cell must check that the centrioles are duplicated and MPF has built up.  Before it is able to leave S phase, it must recognize that all DNA has been replicated faithfully and that there has been no damage accumulation. 

One of the important requirements for S phase to proceed is for sister chromatids to be attached to each other.  This cohesion is facilitated by several proteins named cohesins that loop around both chromatids and “tie” them together.  Smc molecules contain a hinge and ATPase domain:  Smc3 and Smc1 together wrap around the sister chromatids like a cord.  Scc1 and Scc3 act as the latches on the ends of the Smc molecules.  In yeast, the cohesions exist on the chromosome until anaphase and the yeast chromosomes do not become highly condensed.  In mammalian cells, however, the chromosomes become condensed via the condensin complex.  Condensins consist of Smc2 and Smc4 loops along with CAP-G, -H, and –D2.  The condensin complex acts to supercoil DNA.  These sister chromatids, which are connected by the cohesin or condensin complexes are held in place by kinetochore microtubules.  These microtubules emanate from the centrosome and connect to the sister chromatids via the kinetochore.  Additionally, the cell contains astral microtubules that connect the centrosomes to the cell periphery.  Polar microtubules connect to each other near the metaphase plate and act to push on each other, thereby separating the two poles of the cell.

When the cell is in metaphase and the sister chromatids must all align on the metaphase plate, the kinetochore microtubules pull on the chromatids.  These chromatids then align by moving back and forth until they have reached a point where the tension is equal in both directions.  Chromatids that are not attached to kinetochore microtubules actively signal (beep) that the sister chromatids are not prepared for separation (in anaphase).

When the cell is ready to split, its DNA content is store in the nucleus, which is surrounded by the nuclear envelope, made of lamins.  MPF phosphorylates these lamins to disrupt the lamina.  When phosphorylated, the lamins vesiculates and dissociate.  Additionally, nuclear pore complexes dissociate as they, too, are phosphorylated.  This allows for the duplicated DNA to separate to the separate poles of the two daughter cells.

As mentioned previously, Cdc25 is the activating phosphatase for Cdk/cyclin.  During interphase, it is phosphorylated on serine 216 and bound to a 14-3-3 protein.  This protein blocks Cdc25’s NLS (nuclear localization sequence) and exposes its NES (nuclear export sequence), causing Cdc25 to be cytoplasmic.  During mitosis, the phosphorylation of Cdc25 changes and 14-3-3 no longer blocks.  This change exposes the NES, and Cdc25 is phosphorylated by Pin1 to upregulate its activity.  This drives the activation of Cdc2/cyclin B, which is active during mitosis only.