Topoisomerases are a class of DNA-binding proteins that relax DNA supercoils during Transcription, Translation and Replication.

This first view is the basic Topoisomerase IB found in Humans. The purple wound structure inside of the spacefilled protein represents DNA. Human Topo IB is comprised of 765 amino acids when active in vivo, yet there are only crystal structures of the 203 - 765 amino acids. The DNA shown in the all the views is taken from the Tetrahymena thermophilus bacteria.

With the DNA removed, and the protein displayed as a cartoon of its secondary structures, you can see the large center region that accepts the double-stranded DNA while supercoiling is building up.

There are several active amino acids; Arg 488, Arg 590 and His632 bunched together that assist Tyrosine723 in the actual binding of DNA. These amino acids are shown in green. Another amino acid that binds to the DNA, Arg 316, is shown in white. Arg316 is important for DNA supercoil relaxation through molecular-binding interactions during DNA rotation. These amino acids help stabilize the DNA helix when it is being cut.

Tyrosine 723 is the catalytic amino acid of the protein, shown in blue. Tyr723 makes a nucleophilic attack on the phosphodiester bond of the DNA backbone, and breaks the O-P-O bond.

The new bond formed can be seen HERE in a Chime format. The bond is formed between the Oxygen off of Tyr723's Phenyl-OH group and the Phosphate located on the DNA backbone. The Hydrogen bonds, shown as green lines, represent the possible interactions between the assisting amino acids Arg488 and Arg590, as well as some structural support from His632 to Arg590. However, there is more to the tale than this view shows!!

As shown HERE, the His632 actually creates a Hydrogen bond to the scissile oxygen (O2) and sacrifices a hydrogen to the O5 in order for the Tyr723 to make its nucleophilic attack. Once Tyrosine goes through the transesterification reaction with the 3'-O, the protein is considered covalently linked to the DNA at the 5'-end. The helper amino acids, Arg488 and Arg590, make Hydrogen bonds to non-binding Oxygens on the scissile strand to stabilize the newly broken DNA. These interactions allow the DNA to revolve around the non-broken strand and release the supercoiling effects by a process called "controlled rotation."

CONTROLLED ROTATION is the most current theory of supercoiled DNA relaxation. It has been shown that the DNA does not freely spin when attached to the protein, except when the N-terminal domain is removed in-vitro, and has ionic interactions with 5 or 6 positively charged regions that exist downstream of the +1 DNA-Tyr723 binding site.

The DNA is allowed to spin inside the topoisomerase protein, while being stabilized by amino acid residues on the N-terminus portion of the holoenzyme.

Although the N-terminus is not required in-vitro for enzymatic activity, in-vivo studies have found that the N-terminus region controls the speed and ability of rotation.

For more information on the Supercoiling of DNA, CLICK HERE

Protein Opening for DNA Introduction

Using E.coli bacterial topoisomerase I as a model, we can visualize the opening of the protein structure to allow DNA to get inside of it. E.coli topoI is slightlly different in structure, catalytic activity, and energy requirements, but for the purpose of viewing common motifs, it will substitute well.

(1) DNA lines itself up next to the topoisomerase enzyme, as shown here. The green amino acid shown represents the Pro431 present in human topoIB that was discussed above. The blue amino acid represents the Active site Tyrosine723.

(2) In the open conformation, the salt bridge between the "lips" of the protein (between subdomains I and III) have broken apart the DNA is ready to be taken inside the protein. The "hinge" mechanism opened the structure completely and now the DNA can get close enough to the Tyr723 and other catalytic amino acids to be bound.

(3) The DNA has entered the protein structure, however, no binding has occured at this point.

(4) The final step in the Protein-DNA binding is the closing of the structure around the DNA in a "clamp" motif.

Protein Structural Components

Another view of the protein divided by Subdomains

The Human Topoisomerase IB protein is a single chain polypeptide, however, each of the domains, and subdomains, have specific uses during supercoil relaxation and DNA binding.

First, the "Core" Domain (residues 215-636) is broken into four subdomains, labeled I, II, III, and "cap," because of their relative positions in the structure of the protein. The most important amino acids for the binding of DNA are located within subdomain III. The Core domain is crucial for actual enzymatic activity.

Subdomain I (yellow) contains residues 215 to 232 and 320 to 433. The secondary structure, as shown, consists of 1-alpha helix and 2-beta sheets. Although this region does not bind to DNA directly, it does contain several positively charged amino acids on the alpha helix which work for the "controlled rotation" model. In addition, the "hinge" mechanism is located at the connection between subdomain I and III. The actual "hinge" itself is Pro431, which goes through a 120 degree shif to create the "open" structure that allows DNA to get inside the "clamp."

Subdomain II (blue) contains four alpha helicies and only one small beta sheet structure. Together with subdomain I, the "cap" region is created and the connections between I and II subdomains create a V-structure of alpha helicies. Within this V-structure lies a DNA binding amino acid, Arg316, which helps to stablize the broken DNA strand with h-bonding.

Directly following the V- shaped helicies is a region known as the "lips" region. Shown in purple, , this region holds the "cap" to the subdomain III by a salt bridge. This bridge is broken when DNA comes in contact with the protein to cause an "open conformation" that can be viewed below:

Subdomain III (red) serves as the bottom half of the "clamp" of the protein. It consists of six alpha-helicies and 3 beta-sheets. The majority of the catalytic amino acids are present in subdomain III because of its folded position relative to the active site Tyrosine723.

The Core domain works in conjunction with the COOH-terminal domain

(residues 713-765) to bind the DNA double helix. The active site Tyr723

appears on the C-terminal region and is crucial for any kind of activity.

The next region of structural importance is the Linker region (residues 636-713), which contains some positively charged amino acids which causes a restriction in the rotation of the negatively charged DNA. This region, although very cool looking, does not have a lot of functionality that can be described, except for the presence of several charged amino acids. The structure of the Linker is known as a "coiled-coil" structure and is well known in the DNA binding proteins. The linker domain is often removed when reconstituting the enzyme for crystallographic imaging.

The final region, not shown in the crystal structure, or domain cartoon, is the N-terminal domain. The NH2-domain (residues 1-215) is missing because it is a highly charged regoin and the structure does not order very well during the crystal making process. It does not appear in the "Domain" set up because it is not necessary for protein activity in-vivo or in-vitro, however, it does regulate protein activity, and DNA rotation, in both cases. The NH2-domain functions as a control mechanism for the protein in-vivo by serving as a region for protein-protein interactions. Proteins that are especially important binders to the N-terminal domain are p53, SV40, WRN protein, and several transcription factors. These proteins regulate the activity of the topoisomerase, and have possible cancer-target possibilities associated with them. In addition to being a protein interactive domain, the N-terminus also serves to regulate DNA rotation when bound to the Core enzyme due to positively charged amino acids that tend to line the DNA facing portion of the molecule.

Conclusion

Human Topoisomerase IB is a crucial protein in the relaxation of DNA supercoils. Without this protein to relax the DNA during transciption and other DNA opening reations, the DNA would explode .

Topoisomersase IB is able to relax both positive and negative supercoils, which makes it different than the other topoisomerases. It can do this because of the binding of Arg316 and the assistance of His632, Arg590, Arg488, as well as a few downstream amino acids Lys650 and Arg708. The DNA is allowed to rotate within the protein, but not completely freely. It spins around the un-broken DNA strand very carefully, as it is interacted with ionically by the amino acids listed above, and a few other positively charged amino acids.

Helpful Links

Good E.coli TopoI website

Rick's DNA Helicase

References