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.

Cell Cycle I: Cdks and cyclins, OH MY

The Cell Cycle

Understanding the cell cycle is critical to comprehending the many other processes that occur.  A myridad of events, such as DNA damage or cell-to-cell signaling, will affect the frequency of a cell’s division, and, as well, the cell cycle will also impact several pathways within the cell.

DNA replication is central to the cell cycle as well, as it must occur once only during the cell’s S phase.  However, before it can do this, the cell must receive growth signals, consider its size and nutrient availability, and determine if there is any DNA damage.  Several checkpoints have been built into the cell cycle prior to S phase to prevent the cell from unnecessarily replicating its DNA, which is a very costly process energetically.   Thus, the cell would not want to waste its resources if it cannot complete replication or if the DNA is damage.  After all, what would be the use of replicating if the cell’s DNA is damaged and progeny cells may not even survive?  Additionally, synthesis of the DNA must also be coordinated with replication machinery that will actually perform the reactions necessary.  Finally, regulators of replication initiation complexes (see notes on DNA replication for information about initiation complexes) must be phosphorylated or synthesized to prepare the DNA for S phase.

Much of the original research into the cell cycle took place in yeast, either of the budding or fission variety (S. cerevisiae or S. pombe, respectively), due to the ease of identifying mutants that were unable to progress through the cell cycle.  Due to yeast’s morphology, one is able to easily decipher which stage of the cell cycle it is in.  For example, in budding yeast, one is able to tell that the cell is in S phase when it has just begun to schmoo and M phase when its chromosomes are segregated.  As mentioned, the original studies in cell cycle considered mutants that were unable to complete the cycle and arrested at different stages.  This was performed by using temperature-sensitive (ts) mutants of the yeast.

Isolating temperature-sensitive cell cycle mutants simplified:

1.      Mutagenize with your favorite mutagen

2.      Screen for temperature sensitive mutants by replica plating

3.      Look for cells that arrest at a uniform stage of the cell cycle

4.      Sort mutants into complementation groups

5.      Transform mutants with plasmid library to identify gene of interest

Microscopically, it is relatively easy to determine when a temperature-sensitive mutant arrested in the cell cycle due to the distinct morphology.  Cells that arrest in the same phase of the cycle look the same, regardless of whether there are nutrients present or not.  For example, if a cell had arrested in metaphase in mitosis, all of the cells would appear to be schmooing with their chromosomes lined up at the metaphase plate when they are placed at the non-permissive temperature (36^o).  When grown at the permissive temperature (25^o), the cells will continue the cell cycle and will not be synchronized.

The molecular basis of cell cycle control

A great number of molecules are involved in the cell cycle and its regulation.  However, the main complex that is the driving force of the cell cycle is Cdk-cyclin. Cdk, or cyclin-dependent kinase, phosphorylates a number of targets when it becomes activated, and it is only active when it is bound to cyclin, which is a short-lived protein in the cell that is present only during cell division.  In fact, there are two types of cyclins (in yeast): S- and M-cyclin, for synthesis and mitosis cyclins.  Their different binding affinities and abundances during different stages of the cell cycle give Cdk its specificity.  Cdk-cyclin can also be called MPF, for mitosis-promoting factor, when it is the engine driving the G2-to-M transition, or SPF, for S-phase-promoting factor, when used for the G1-to-S transition. 

Cyclin binding isn’t the only requirement for Cdk to become active.  In fact, there are a number of additional steps that must occur for the enzyme to phosphorylate its targets.  Two of the first proteins identified that affects Cdk’s activity were Cdc25 and Wee1.  Wee1, when expressed in excess, led to elongated  yeast cells; while Cdc25 excess led to small cells (cells that passed through mitosis quickly).  Thus, Wee1 acts to inhibit MPF (Cdk-M-cyclin) and Cdc25 activates it.  The exact mechanisms was found to be phosphorylation and dephosphorylation:  Wee1, a kinase, adds an inhibitory phosphate to Cdk on tyrosine 15; Cdc25 phosphatase removes this inhibitory kinase and promotes progression through the cell cycle.  Cdk required both the removal of the inhibitory phosphate and the binding of cyclin to become partially active, but this, too, is not all for a fully active enzyme.  In addition, Cdk must be phosphorylated by a Cdk-activating kinase (CAK), which adds an activating phosphate group to make the enzyme fully active.  This phosphorylation event at threonine 161 on Cdk results in the extension of the T-loop to allow substrate binding and facilitate catalysis.

Due to the importance of Cdk-cyclin for the cell, it has several layers of regulation, and there are many other factors other than phosphorylation, as described above, that regulate it.  When Cdk-cyclin is associated with a CDK inhibitor (CKI) such as p27 (mammalian cells) or Sic1 (yeast), its activity is blocked.  Another CKI is p21, which is involved in DNA damage response.  When DNA damage is detected and p53 is activated, p21 is active and binds Cdk/cyclin to prevent cell cycle progression. CKIs are deactivated by SCF, which bind and ubiquitinated CKI when it is phosphorylated (CKI acts as a phosphordegron – it is targeted for degradation upon phosphorylation).  When SCF is active and ubiquitinates the CKI, Cdk-cyclin can become active.  Sic1 in yeast is involved in binding the S-phase cyclin and Cdc28 (the Cdk), and it is phosphorylated by the Cdk-cyclin present during G₁.  In this way, the Cdk-cyclin that is active prior to S-phase cyclin/Cdc28 works to activate the next stage in the cell cycle.

Review: Regulation of Cdk/cyclin

·         Cyclin must bind Cdk for activity

·         Activating phosphate (added by CAK)

·         Inactivating phosphate (added by CKI/Wee1; removed by Cdc25)

·         Inactivation by p21/27

·         p21 is stimulated by p53 during DNA damage

Cyclin D is an important cyclin molecule involved in cell cycle progression that binds Cdk4/6.  When this complex is active, the Rb protein is phosphorylated.  Normally, Rb will be found to E2F, an important transcription factor.  When Rb is phosphorylated, it dissociates from E2F, which then acts as a transcriptional activator to upregulate transcription of Cyclin E / Cdk2 as well as its own transcription and much of the replication machinery.

The cyclincs and Cdks of mammals:

Stage	Cdk	Cyclin
G1	Cdk4	Cyclin D
	Cdk6	Cyclin D
G1 – S	Cdk2	Cyclin E
S	Cdk2	Cyclin A
G2 – M	Cdc2 (Cdk1)	Cyclin B

DNA Repair 5: The final chapter

Another method of DSB repair is via non-homologous end joining (NHEJ), which seems significantly riskier for the cell because it involves the ligation of two ends of DNA.  Nonetheless, it appears that this is the preferred mode of double-stranded breaks and is not random.  Hypotheses have been considered that the proteins involved in NHEJ are very abundant in the cell, and when DSB occur, they are immediately available to attach to the DNA and ligate it back together.  This process is performed by Ku heterodimers, which are proteins that bind directly to the ends at the DSBs.  Ku proteins then recruit DNA-PKcs (DNA protein kinases), which, in turn, recruit Artemis endonuclease to open the ends and remove any hairpin structures that may have developed.  The ends of the DNA are then slightly chewed away, polymerized, phosphorylated, and ligated together.  The action of XRCC4, another protein that complexes during NHEJ, and Ku is used to recruit DNA ligase IV.  The final product of this process is a DNA strand that is very similar to the original DNA, but it is not exact:  NHEJ does result in mutated DNA sequences.

An excellent review on DSBs and their repair can be found here.

Double-stranded breaks sound like a terrible thing to have happen to a cell.  After all, when DNA breaks and is repaired, there is a high chance that the mutation that arises will be harmful to the cell.  Nonetheless, double-stranded breaks aren’t detrimental to the cell, and they are even required for certain normal cellular processes.  For example, in the development of immunoglobulins, lymphocytes’ genomes are rearranged and parts are removed in a process termed VDJ recombination.  This allows for the lymphocytes to increase diversity in the coding regions of the immunoglobulin genes and helps to protect us from disease.  To begin VDJ recombination, site-specific endonucleases RAG-1 and RAG-2 induce DSBs and repair these breaks via NHEJ.

As mentioned above, Ku proteins bind directly to DSBs when they occur, but how do they know when a DSB occurs?  The detection of these breaks is performed by the MRN complex composed of Mre1, Nbs1, and Rad50.  Mre11 acts as an endonuclease, Rad50 as an ATPase, and Nbs1 as a scaffold.  These proteins, once in complex, hold the DNA together and recruit other proteins, resulting in several signaling cascades.  These signaling cascades have been studied in detail using ionizing radiation induced foci (IRIF) in vitro.  The data that has been gathered from these studies shows that the MRN complex recruits ATM, a PI3K-family kinase, that becomes active during DSB formation.  ATM then phosphorylates H2AX, an H2A variant, to form γH2AX.  γH2AX acts as a scaffold to recruit more ATM and many other proteins, such as chromatin remodeling complexes, to the site of the lesion.  The cell cycle is arrested via phosphorylation of Chk2 by ATM (or phosphorylation of Chk1 by ATR – ATM-related kinase).  Chk1 and Chk2 phosphorylate CDC25 phosphatases and inactivate them, which prevents the activation of cyclin-dependent kinases.  Additionally, ATM can induce phosphorylation and activation of p53, leading to G1/S arrest.  Tip60 is another protein involved in DNA repair and is involved in acetylating ATM to activate it.  ATM and Tip60 exist in a constitutive complex that is only activated on DNA damage signaling.  MCD1 acts as a scaffold that binds phosphorylated H2AX and docks ATM/Tip60.

DSB detection simplified:

1. The MRN complex recognizes and binds the free ends of the DNA
2. ATM is recruited to the foci
3. ATM phosphorylates H2AX, Chk2 and p53
4. Chk2 phosphorylates CDC25
5. Cell cycle is arrested and chromatin opens for DNA repair

Several diseases are associated with defects in the DSB response pathway.  Among them are ataxia-telangiectasia, resulting from mutations in ATM.  Several mutations in ATM will result in the A-T phenotype, and approximately 1% of the population carries one mutated allele.  Those that suffer from this syndrome show immunodeficiency and increased cancer rates, and they have a life expectancy of 17-23 years.  In addition to A-T, Nijimegen breakage syndrome results from mutations in Nbs1, and A-T like disorder (ATLD) results from Mre11 mutations.  Breast cancer may also have connections to DSB repair, as BRCA1 is associated with the BASC complex, which is involved in this and many other pathways for DNA repair.  BRCA1 acts as an ATM substrate and localizes to IRIFs, possibly acting as a scaffold.

More information about ataxia-telangiectasia can be found here.

On NBS can be found here.

A paper about ATLD: here.

Identification of genes involved in DNA repair

A number fo methods have been developed that specifically scan organisms in order to detect defects in DNA repair.  Isolation of the affected gene and protein then allows for recognition of specific DNA repair proteins.  Specifically, one can look at the frequency of mutations (see Ames test, for example) as well as elevated sensitivity to ultra-violet or ionizing radiation.  Cell cycle arrest (cell division defects) and apoptosis are common phenotypes associated with defective enzymes as well.

DNA Repair 4

A further method of repairing small lesions in the DNA is via mismatch repair (MMR), which fixes errors in replication and heteroduplexes from recombination intermediates.  The process involves the Mut proteins in prokaryotes.  MutS bound to ATP recognizes and binds the unpaired base to activate the mismatch repair enzymes.  MutH is involved in determining which DNA strand is methylated, indicating the parent strand (the correct copy of the DNA that should be used to repolymerize the incorrect copy).  MutL facilitates in assembling the MMR complex, which consists of helicases and exonucleases.  Repair is then done by DNA polymerase and ligase, and methylation of the newly copied (and unmutated) strand is then performed.  MutL and MutS homologues are found in both yeast and humans, with varying roles and functioning as heterodimers.  Additionally, in eukaryotic MMR, the ability of the enzymes to distinguish between parent and daughter strands is not as clear.  Hypotheses have emerged that consider interaction with PCNA and identification of nicked strands (on the nascent DNA) as mechanisms that may assist in determining which strand needs to be repaired.

 

One consequence of an impaired mismatch repair system is microsatellite instability, a variation in tandem repeat sequences.  In cells both with and without a functioning MMR system, changes in the number of repeats is commonly observed as the DNA is being replicated.  This variation is due to slippage of DNA polymerase as it runs through repeated sequences, as if it has gotten lost along the way.  Usually, the microsatellite instability is held in check by MMR enzymes that are able to recognize the bubble that forms in the DNA.  However, in those individuals with mutations, there can be a vast array of repeat numbers in these microsatellites, up to 3.3x10^-3 mutations per cell per generation.   

The most catastrophic event that can occur in a cell is double-stranded breaks (DSBs) in the DNA, which can be caused by radiation and mutagens, but it can also result from DNA replication and physiological processes (harmful metabolites that are not controlled by the cell properly).  Two methods have evolved to prevent these DSBs from having a negative impact on the cell, which we will consider now.

The first method of DSB repair is by homologous recombination.  (Details about this pathway can be found in additional posts about recombination.)  There are two methods for recombination: synthesis-dependent strand annealing and single-strand annealing.  In synthesis-dependent strand annealing homologous recombination, strand invasion of the undamaged DNA (sister chromosome) by the DNA that contains the double-stranded break.  New DNA is synthesized from the correct copy and ligated to remove the DSB.  In contrast to synthesis-dependent annealing, single-strand annealing relies on an uneven breakage in the DNA.  Several bases on each side of the DNA strand are removed, and the staggered ends are annealed to create fixed DNA.  This method of DSB repair is especially common in stretches of repeated-sequence DNA.

DNA Repair 3

Mechanisms of Repair: NER and BER

In the case of damaged DNA, be it via radiation, oxidation, or incorrect basepairing, it will recognize the mutation via several different mechanisms and will begin to repair the error, if it can. 

Nucleotide excision repair is one common mechanism in which the cell removes a number of nucleotides surrounding a DNA lesion and “fills in the gaps.”  This repair mechanism is frequently in use because it is used to repair damage from UV radiation, especially thymine dimers.  To accomplish this, the lesion is first detected by XPC and HR23.  Next, XPB and XPD, complexed with TFIIH, unwind the DNA via helicase activity.  Then the entire base and ribose-phosphate backbone is removed in a span of 24-32 nucleotides by XPC and ERCC1-XPF, which act as endonucleases.  DNA polymerase and ligase then replace the missing nucleotides and close the DNA.

NER simplified

1. Detection of lesion by XPC and HR23
2. XPB, XPD, and TFIIH bind and act as helicases
3. XPC and ERCC1-XPF act as endonucleases to remove 24-32 nucleotides
4. Replacement and ligation of DNA by DNA pol and ligase

An additional pathway involves the binding of CSA and CSB to the lesion when RNA polymerase II is actively transcribing the DNA into mRNA.  CSA and CSB then recruit the same proteins as described above and help in DNA repair. 

Diseases associated with defects in these two pathways can have severe phenotypes.  Xeroderma pigmentosum manifests itself as extreme sensitivity to UV light with an increased risk of skin cancer.  Seven genes have been found to be involved in the disease phenotype and can be associated with different severities of the disease.  An excellent review of XP can be found here. Additionally, Cockayne syndrome is associated with defects in the CSA/CSB pathway.  More information about this disease can be found here.

Similar to nucleotide excision repair, base excision repair fixes small lesions in DNA, but instead of using endonucleases to cleave small sequences of DNA, base excision repair removes single bases, not the entire nucleotide.  The incorrect base often takes the form of uracil, which can be accidentally incorporated in place of dTTP or via the deamination of cytosine.  While the incorporation of dUTP instead of dTTP is not mutagenic (A will still bind either T or U), deamination of cytosine can lead to a conversion of a C-G pair to a T-A pair. 

To repair via base excision repair, DNA glycosylases flip the incorrect uracil base to stick outside the DNA backbone.  AP endonuclease (APE) then forms a nick at the site of the lesion and removes the sugar phosphate (via lyase).  The missing base is then replaced by DNA polymerase β and the nick is sealed by DNA ligase.  The two DNA glycosylases (UDG: uracil DNA glycosylases) of mammals follow the replication fork and travel the DNA to find incorrect uracil bases.

Base excision repair simplified

1. Uracil DNA glycosylase (UDG) recognizes uracil and flips the base
2. AP endonuclease (APE) nicks the site of the uracil
3. Lyase removes the sugar phosphate
4. Replacement of base by DNA polymerase β
5. Ligation of DNA by ligase

DNA Repair 2

Avoiding Permanent DNA Damage

When a cell encounters DNA damage, it has a number of responses that help to correct any errors and propagating these errors to progeny cells.  Thus, the cell devotes a number of enzymes to detecting and repairing any lesions in the DNA.  Correcting DNA lesions is more important than fixing errors in transcription or translation because of the relative lifespans of the DNA versus mRNA and protein:  mRNA and protein are both expressed and, while they can be longlived, they are not passed on to progeny.  In contrast, DNA of daughter cells is an exact copy of the mother cell, and these daughter cells will carry any lesions that the mother cell carried as well.  Therefore, to prevent long-lasting mistakes in the DNA, the cell fixes any errors that it may encounter.

One of the first hurdles that the cell encounters when it must maintain its genome is that it must accurately copy it, but it must also perform polymerization of DNA quickly enough to facilitate survival.  Once in a while, DNA polymerase will make a mistake, roughly one in a billion base pairs is misincorporated, which is an astoundingly low rate.  Such a low mutation rate is thanks, in part, to the 3’-to-5’ exonuclease activity of DNA polymerase.  When the enzyme is polymerizing DNA and it accidentally adds the wrong nucleotide, this mistake is detected because DNA polymerase is not able to polymerize efficiently from a misincorporated nucleotide.  When the mistake is detected, the exonucleolytic activity of DNA polymerase kicks in and removes several nucleotides behind the incorrect base.  Then, it reverts to its normal function and continues to replicate the DNA. 

When DNA polymerase δ is replicating the DNA and there is an identified lesion ahead of the replication fork, the cell can choose to bypass the lesion and repair it later.  Such a scenario is called translesion synthesis, which uses an alternate polymerase and is not high fidelity, as DNA polymerase δ.  The process involves the ubiquitination of PCNA (proliferating cell nuclear antigen) and the involvement of a number of factors normally involved in DNA repair, including RAD6 proteins and DNA polymerase η.  More information about this alternative mechanism can be found here. 

DNA Repair 1

Causes of DNA Damage

Constantly, day-in and day-out, the genome is assaulted by a number of forces that lead to damage and mistakes that can lead to severe problems for the organism under the onslaught.  Included in the forces that cause the DNA damage are chemicals, radiation, and especially reactive metabolites (reactive oxygen species, for example).  Such mutagenic agents can lead to damage in the form of thymidine dimers, single-strand breaks, creation of abasic sites, or the formation of double-stranded breaks.  Specifically, UV radiation induces pyrimidine dimers, while ionizing radiation can cause double-stranded breaks.  Toxic chemicals can be mutagenic in themselves, or they can be activated by cellular metabolism into mutagenic chemicals.  In addition, DNA replication errors and the inherent mobile genetic elements can lead to genetic mutation.

Some of the most common mutations in DNA occur via hydrolysis a nucleotide base.  Due to the high concentration of water within the cell, the DNA is constantly exposed to the harmful effects of water.  Depurination results in a nucleotide without its guanine or adenine base.  Deamination is a base conversion event in which a methylated cytosine residue to converted to thymine.  Such an event can be especially dangerous to the cell because it can induce base changes if the mutation is not fixed immediately. 

Chemicals that induce mutations come in several forms: base analogs, intercalating agents, and base modifiers.  In the case of base analogs, the chemicals resemble that of the natural bases found in the genome but have altered binding potential, leading to the misincorporation of bases that fit improperly.  Examples of base analogs include 5-bromouracil (5-BU) and 2-aminopurine (2-AP).  Intercalating agents do not pair with the DNA bases, but they affect the structure of the DNA helix during DNA synthesis, leading to improper pairing and the induction of mutations.  Examples of intercalating agents include proflavin and acridine orange.  When a base is modified, it can also affect the DNA structure, leading to altered pairing and the induction of mutations.  Chemicals that modify bases as alkylating agents include ethyl methanesulfonate (EMS) and nitrosoguanidine (NG). 

Testing for mutagenic activity

In order to examine whether a chemical is mutagenic in a living system, using the model organism Salmonella, the Ames test is employed.  First, mice are exposed to the mutagen and injected with arochlor to induce hepatic function, where most chemical mutagens are activated.  Then, liver extract is homogenized and added to Salmonella.  The Salmonella strain used contains a His- marker, making it unable to grow in the absence of histidine.  The ability for the strain to grow on media lacking histidine works as a readout for the mutagenic capability of the chemical:  the most colonies that grow on the selective media, the more mutants were able to convert to His⁺ and the higher the mutagenic capacity of the tested chemical.  Extensions of the Ames test have been developed to specifically look at base substitutions and insertions or deletions.

The Ames Test Simplified:

1. Expose mouse to potential mutagen
2. Inject mouse with arochlor to induce hepatic function
3. Isolate and homogenize liver
4. Combine homogenized liver with Salmonella typhimurium (His^-)
5. Plate Salmonella on agar lacking histidine
6. Count number of colonies that have grown on the plate as a measure of mutagenicity.

More information about the Ames test can be found here and here.

Note: Links provided in this post are only a sampling of the vast amount of information available about these chemicals and processes.