Structural Biochemistry/Nucleic Acid/DNA/Supercoiling and Nucleosomes
The structure of DNA does not only exist as secondary structures such as double helices, but it can fold up on itself to form tertiary structures by supercoiling. Supercoiling allows for the compact packing of circular DNA. Circular DNA still exists as a double helix, but is considered a closed molecule because it is connected in a circular form. A superhelix is formed when the double helix is further coiled around an axis and crosses itself. Supercoiling not only allows for a compact form of DNA, but the extent of coiling also affects the DNA’s interactions with other molecules by determining the ability of the double helix to unwind.
Although the supercoiling provides an organized way to tightly compact DNA, the structure is relatively unstable as a result of torsional strain. In order to minimize the energy required to maintain the structure, the number of twists and writhes are minimized. Twists refer to the number of turns the double helix makes around the superhelical axis. Writhes refer to the circular distortion, bending, and overall non-planarity of the DNA strand.
Supercoiling changes the shape of DNA. The benefit of a supercoiled DNA molecule is its compactibility. In comparison to a relaxed DNA molecule of the same length, a supercoiled DNA is more compact. How this is reflected in experimentation is that supercoiled DNA moves faster than relaxed DNA. Therefore, the structural differences can be analyzed in techniques such as electrophoresis and centrifugation.
Positive and Negative Supercoilings
1. Negative supercoiling is the left-handed coiling of DNA thus winding occurs in the counterclockwise direction. It is also known as the "underwinding" of DNA.
2. Positive supercoiling is the right-handed, coiling of DNA thus winding occurs in the clockwise direction. This process is also known as the "overwinding" of DNA. (CORRECTION FIXED on 10/23/17 - DV, original error had the negative and positive supercoiling definitions reversed. Also provided more basic clarity to supercoiling).
Although the helix is underwound and has low twisting stress, negative supercoil's knot has high twisting stress. Prokaryotes and Eukaryotes usually have negative supercoiled DNA. Negative supercoiling is naturally prevalent because negative supercoiling prepares the molecule for processes that require separation of the DNA strands. For example, negative supercoiling would be advantageous in replication because it is easier to unwind whereas positive supercoiling is more condensed and would make separation difficult.
Topoisomerases unwind helix to do DNA transcription and DNA replication. After the proteins have been made,the DNA template supercoils by the force to make chromatin. RNA polymerase also influence DNA strand to have two different supercoiled directions. The region RNA polymerase has passed forms negative supercoil while the region RNA polymerase that have not passed forms positive supercoil. By these processes, supercoils are generated.
Topoisomerases are enzymes that are responsible for the introduction and elimation of supercoils. Positive and negative supercoils require two different topoisomerases. This prevents the distortion of DNA by the specificity of the topoisomerases.The two classes of topoisomerases are Type I and Type II. Type I stimulates the relaxation of supercoiled DNA and Type II uses the energy from ATP hydrolysis to add negative supercoils to DNA. Both of these classes of topoisomerases have important roles in DNA transcription, DNA replication, and recombinant DNA.
Topoisomerase form loops (unwinded regions of the double helix) of negative supercoils. If the DNA lacks superhelical tension, there is no unwinding of supercoils.
Type I topoisomerase
Type I topoisomerase act by creating transient single-strand breaks in DNA. This is further classified as type IA and type IB.
Type IA topoisomerases
Type IA topoisomerases enzyme is a 695-residue monomer and it relaxes negatively supercoiled DNA. First, Type IA cuts a single stranded DNA and catenates two circles of single stranded DNA. Then it unwinds the supercoiled duplex DNA by one turn. Type IA has a specific strand-passage mechanism which is the denaturation of type IA incubated with single stranded DNA that yields a linear DNA by phospho-Tyr diester linkage.
Type IB topoisomerases
Type IB mediates a controlled rotation mechanism to relax both negative and positive supercoils. Type IB cleaves a single strand of a duplex DNA through the nucleophilic attack of an active site with Tyr on a DNA to yield a 3'-linked phospho-Tyr intermediate with 5'-OH group. Type IB consists of several domains and subdomains. Interestingly, type IA topoisomerases form a covalent intermediate with the 5' end of DNA, while the IB topoisomerases form a covalent intermediate with the 3' end of DNA. Historically, type IB topoisomerases were referred to as Eukaryotic Topo I, but Type IB topoisomerases are present in all three kingdoms of life.
Type II topoisomerase
Type II topoisomerase is an enzyme that require ATP hydrolysis to complete a reaction cycle in which two DNA strands are cleaved, duplex DNA is passed through the break and the break is resealed. Type II cuts both strands a DNA double helix, passes another unbroken DNA strand through it, and then reanneals the cut strand. It is also split into two subclasses: type IIA and type IIB topoisomerases, which share similar structure and mechanisms. Examples of type IIA topoisomerases include eukaryotic topo II, E. coli gyrase, and E. coli topo IV. Examples of type IIB topoisomerase include topo VI. Supercoiling requires energy because it is torsionally strained. Thus, through the coupling to ATP hydrolysis it can introduce negative supercoils.
In bacteria, Type II topoisomerase is also known as DNA gyrase. Gyrase is an enzyme that acts similarly to human Type II topoisomerase. Antibiotics act on bacterial enzyme by blocking the binding of ATP to gyrase and thus deactivating the breaking and joining of bacterial DNA chains.
Nucleosomes allow for the compact packing of linear DNA. Nucleosomes are complexes of DNA and histones, consisting of ~145 base pairs of DNA wrapped around in a left-handed superhelix around a histone octomer, which are a group of small proteins. Histones contain a large amount of positively charged amino acids such as lysine and arginine which allow them to bind to the negatively charged DNA molecule. The histone octamer is composed of two copies each of H2A, H2B, H3, and H4. The two loops of DNA around the histone are attached to the histone also using the H1 histone. Nucleosomes are further arranged in a stacked helical complex. Through the extensive wrapping of DNA around the histones, as well is the helical arrangement of the nucleosomes, the linear DNA is able to be compacted. The structural folding of the nucleosomes eventually forms a chromosome.
Chromatin refers to the structure of DNA and its accompanying histones. Chromatin is composed of repeating units called nucleosomes. The five major histones found in chromatin are H2A, H2B, H3, H4, and H1.
In gene clusters, protein genes of histone are present and these are expressed in S phase. Once it is expressed, it forms histone octamers. With interactions of 146 base pairs of DNA double helix, histone octamer becomes a nucleosome. When histones bind to DNA, it is depended on the amino acid sequence of histone, not the nucleotide sequences of DNA.
Histones and Transcription Regulation
Histones always appear to remain attached to the DNA even through transcription. The fact that nucleosomes are able to change shape and position allow for transcription to occur and RNA polymerase to move along the DNA strand. Slight loosening of the binding between the histones and DNA are accomplished by acetylation of the histones, which neutralizes the positively charged residues. Meanwhile, binding is made tighter through methylation to restore the positive charge of the histones. By changing the charge of the histones in this manner, gene transcription can be regulated. Histone Chaperones are proteins that mediate the assembly and disassembly of the chromatin to form correct nucleosomes sequences and aid in stable folding conformations. These proteins function to protect and shield the histones from forming incorrect and unwanted aggregates with DNA because of the high ionic strength that exists between DNA and Histones. DNA is primarily negatively charged molecule and histones are positively charged therefore, there exists a strong affinity for each other. Histone Chaperones, which are positively charged, help to guide histones to form octamers and correctly bind to DNA by shielding and masking the negative charge of DNA. There are different types of histone chaperones, including β- sandwich, α/α earmuff, Β-propeller and β-barrel chaperones. Β-sandwich chaperones are chaperone monomers that form β-sheets with the histones. An example of these types of chaperones is ASF1 or anti-silencing function chaperones involved the overexpression during yeast replication. In addition ASF 1 is the first histone used during assembly of the chromatin. α/α earmuff chaperones are dimers that form α helical conformations of histone/DNA complex. An example would include NAP chaperones which are used to transport histones from the cytoplasm to the nucleus during chromatin assembly. Β-propeller chaperones were the first chaperones to be distinguished using NMR and crystallography techniques. These pentamer chaperones function is the storage of histones. Β-Barrel chaperones are heteroligomers that help facilitate chromatin transcription. In addition, there are irregular or variant histone chaperones that do not fit into any specific structural category. All of these different types of chaperones are involved in different stages of assembly of disassembly of chromatin. The energetics of Chromatin assembly and disassembly are regulated by histone chaperones. Assembly which is an energetically favored process because as the histones bind with DNA it forms a more stable structure causing a decrease in energy. On the other hand, disassembly is an energetically unflavored process needing the use of ATP to break apart the stable histone/ DNA interactions.
Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro.
ATP-Dependent Nucleosome Sliding Mechanism
The paper “Mechanisms of ATP-dependent nucleosome sliding” by Gregory D Bowman, researches how ATPase motors engage and manipulates nucleosomal DNA and discusses possible mechanisms for ATP-dependent sliding of nucleosomes. ATPase motors are shared between chromatin remodelers and collections of different protein machines. The ATPase motor generates torsional strain when it engages with DNA at an internal site on the nucleosome. The torsional strain in the nucleosomal DNA is a result of the ATPase motor acting at SHL2 region. Protection of nucleosomal DNA between SHL2 and the entry/exit site is increased Isw2 ATPase is activated. ATP-dependent crosslinking of the Isw2-subunit Dbp4 to SHL4 promotes hydrolysis-dependent changes. Iswi-type remodelers form template-committed complexes that allow for nucleosomes to slide processively.
Bowman also explains possible variations of the bulge/loop propagation model using ATPase motors. One model suggests that the ATPase motor uses translocase abilities to pull DNA from an entry/exit site in a continuous manner. This pumping allows for a remodeler to create a bulge that would rapidly diffuse to a distant entry/exit site. Another model suggests the histone-DNA contacts are disrupted by a DNA loop that is developed by a remodeler ATPase around the SHL2 region. This disruption pulls DNA for the linker and the ATPase motor would move toward dyad along the DNA loop.
Chromatin remodelers are mainly involved in DNA packaging and facilitating the transcript elongation process. For example, when a DNA strand coils with nucleosomes for packaging into chromatin, chromatin remodelers arrange the nucleosomes in a regular distance for effective condensation of DNA strands. Furthermore, in some processes where nucleosomes have to be modified, chromatin remodelers may disassemble the nucelosome into histones or even detach the whole nucleosome from the DNA. The processes that require nucleosome modification by the chromatin remodeler include DNA repair, recombination, transcription and replication. The following picture displays an example of how a chromatin remodeler may be used during transcription catalyzed by RNA Polymerase II.