| 
  • If you are citizen of an European Union member nation, you may not use this service unless you are at least 16 years old.

  • You already know Dokkio is an AI-powered assistant to organize & manage your digital files & messages. Very soon, Dokkio will support Outlook as well as One Drive. Check it out today!

View
 

Justin-Neese

Page history last edited by Christopher Korey 14 years, 10 months ago

Topoisomerase II


 

Content

 

 I.  Function

     A.  Double strand DNA cuts

     B.  Topology

     C.  Catenation

     D.  Prokaryotes vs. Eukaryotes

 II.  Classification

     A.  Topoisomerase IIA

     B.  Topoisomerase IIB

 III.  Structure

     A.  Topoisomerase IIA

     B.  Topoisomerase IIB

     C.  Comparison of two types (IIA & IIB)

 IV.  Mechanism

     A.  "Two-gate" mechanism

          1.  Gated segment

          2.  Transfer segment

          3.  C-terminal gate

     B.  ATP hydrolysis

     C.  Topo IIA vs. Topo IIB

 V.  Inhibition

    A.  Leads to death of cells

     B.  Inhibitors

     C.  Poisons

VI.  Medical Applications

A.  Antibiotics

     B.  Anticancer Drugs

 


 

 

I. Function:

 

     Topoisomerase II is involved in many DNA processes such as transcription, replication, and catenation (Wang 1996).  Its main function is to manage the topology of DNA by relieving or inducing tension in the DNA helix brought about by supercoiling (Osheroff et al 1991).  This is done by making a double stranded cut in the DNA helix and passing the other strand through this break (Everything2.com).  ATP hydrolysis provides the energy for this process to occur, thus topoisomerase II requires ATP in order to perform its function (Wikipedia).  The DNA strand that is cut is called the gate segment or the "G-segment" and the DNA strand that is passed through that cut is called the transport segment or the  "T-segment" (Holden).  The cut ends of the G-strand are rejoined once the tension is relieved or induced.  By cutting both DNA strands, Topoisomerase II changes the linking number by 2 (Biochemistry 3107 Course).  Supercoils can either be positive or negative depending on the direction the strands are twisted (Ussery 2000).  Positive supercoils are introduced when the number of turns in a DNA helix is increased (Ussery 2000).  Likewise, negative supercoils are introduced when the number of turns is decreased (Ussery 2000).  Topoisomerase II can only add negative supercoils to DNA.  This is done by either relaxing positive supercoils or inducing more negative supercoils.  Keep in mind, that the number of base pairs cannot change for supercoiling to occur.

 

 

Figure 1.

 

     Transcription - During transcription, a portion of DNA is unwound to form a transcription bubble.  According to DNA topology, since the number of turns in the helix is decreased when this occurs, negative supercoils are introduced in the adjacent portions of the DNA as shown in Figure 2 below (Nitiss 2009).  Here both topoisomerase I and topoisomerase II work to relieve the tension created by the opening of the transcription bubble.  Without the work of these two proteins, adjacent DNA would form knots that would hinder transcription (Learning Space).  Both enzymes work together at these adjacent sites to release supercoiling, allowing for smooth, faster transcription of that region of DNA (Learning Space).  

Figure 2. 

 

(Demonstration of Topoisomerase's effect on supercoiling)

YouTube plugin error  

 

     Replication - Topoisomerase II functions in the same manner during DNA replication as it does during transcription. (Previously explained)

 

     Catenation - DNA catenation is the linking of two or more circular DNA rings together, similar to the way a chain is built from clasping rings together (Ip et al 2003).  Likewise, decatenation is the separating of linked rings of circular DNA into individual DNA rings (Open Learn).  

 

                              

              Figure 3.                                                                          Figure 4.

 

 

Prokaryotes vs. Eukaryotes - 

     Prokaryotic and Eukaryotic type II topoisomerases are structurally different.  The biggest difference is in their composition.  Prokaryotic topoisomerase II is a heterodimer (A2B2), meaning that it consists of two dimers of two different subunits (Wikipedia).  Eukaryotic topoisomerase II, however, is a homodimer (A2), meaning that it is made up of one subunit in dimer form (Holden).

 

     Another difference between prokaryotic and eukaryotic type II topoisomerases is the activity level required to perform their function.  Eukaryotic topoisomerase II requires little active effort to carry out its task, while prokaryotic topoisomerase II is highly active in its function (Holden).  The reason for this difference lies in the way eukaryotic DNA is packaged.  Eukaryotic DNA, in order to fit inside the nucleus of a cell, is wrapped around proteins called histones.  This creates numerous positive supercoils within the DNA which help to compact the DNA into their chromosomes (Lutter et al 1992).  This phenomenon is illustrated in the following video animation:

 

(Compacting DNA via supercoiling)

YouTube plugin error

 

The wrapping of DNA around histones causes it to be highly positively supercoiled (McClendon et al 2005).  The potential energy stored in these positive supercoils assist eukaryotic topoisomerase II in its function (Lutter et al 1992).  Because of the highly positively supercoiled nature of eukaryotic DNA, it takes very little effort for eukaryotic topoisomerase II to relax these supercoils (Holden).  In contrast, prokaryotic DNA in its normal state is circular and contains no supercoils.  Without the potential energy stored in positive supercoils, it takes significantly more active effort for prokaryotic topoisomerase II to add negative supercoils to the DNA. 

     The nature of how eukaryotic DNA is stored brings about another factor that distinguishes prokaryotic and eukaryotic topoisomerase II.  Eukaryotic topoisomerase II can only relax positive supercoils (Charvin et al 2003).  It adds negative supercoiling to DNA by relaxing the positive supercoils that are normally present in eukaryotic DNA (McClendon et al 2005).  Without the presence of positive supercoils, eukaryotic topoisomerase II cannot perform its function.  However, because of the nature in which eukaryotic DNA exists, eukaryotic topoisomerase II does not need to be able to induce negative supercoils on its own for it to be useful to the cell.  In contrast, prokaryotic topoisomerase II can add negative supercoils to DNA regardless of pre-existing supercoils (Crisona et al 2006).  It can relieve tension in DNA by relaxing positive supercoils, or it can induce more tension by adding negative supercoils to an already negatively supercoiled DNA (Charvin et al 2003). 

 

 


 

 

II. Classification:

 

     Two subclasses of topoisomerase II exist.  These are topoisomerase IIA and topoisomerase IIB (Wikipedia).  These two types of topoisomerase II are both structurally and biochemically different from one another.

 

Topoisomerase IIA - These types of topoisomerase II are the more prevalent of the two subclasses.  It encompasses a much wider span of life and includes enzymes such as eukaryotic topoisomerase II, prokaryotic topoisomerase IV, and DNA gyrase (Wikipedia).  This type of topoisomerase II is essential for life in most organisms. 

 

Topoisomerase IIB - This subclass is found in archaea and some plants (Wikipedia).  It is structurally and biochemically unique from any other subclass of topoisomerase II and includes only one member, topoisomerase VI (Wikipedia).

 

 


 

 

III. Structure:

 

Topoisomerase IIA - Enzymes that fall under this category have many different structures but are all made up of a few important motifs.  These include the ATPase domain at the N-terminus, the Toprim domain, the DNA-binding core, and a C-terminal domain (Schultz et al 1996). 

 

          Prokaryotes: Topoisomerase IV in E. coli, a type of topoisomerase IIA, will be used as an example to demonstrate the general structure of type IIA topoisomerases.  Topoisomerase IV is a tetrameric heterodimer made up of two E subunits (ParE) and two C subunits (ParC) (Holden).  Figure 5 below shows how the ParC (light blue) and the ParE (dark blue) subunits are arranged to form this protein.  The ATPase domains are shown in yellow and each tyrosine active site is represented by a red "x" on the ParC subunits.  Figure 6 shows the crystalline ribbon structure of these subunits.

                              Figure 5.                          Figure 6.

 

               DNA-binding Core:  This domain binds to the G-segment DNA and holds it in place.  When ATP is hydrolized, the DNA-binding core cuts the G-segment into two peices, creating a gate through which the T-segment DNA will eventually pass (Schultz et al 1996).  The CAP domain, also referred to as the winged helix domain, is located on the DNA-binding core and contains the catalytic tyrosine active sites (Berger et al 1998).  Figure 7 shows where the CAP domain is located on the ParC subunits.  This CAP domain works with the toprim domain to form the cleavage complex.

                         Figure 7.

 

               Toprim Domain:  The toprim (topoisomerase-primase) domain works together with the CAP domain to form the cleavage complex (Berger et al 1998).  Its flexibility facilitates this interaction.  The toprim and CAP domains are responsible for allowing the binding and cleavage of the G-segment to be executed separately as an uncoupled process.  The toprim domain and the DNA-binding core together make up the central core (Wikipedia).

 

               ATPase N-terminal Domains:  These domains are located on the two B subunits (shown in dark blue in Figure 5) and dimerize in the presence of ATP due to their ATPase activity to create a closed formation (Berg et al 2002).  This closed formation "captures" the T-strand of DNA and passes it through the G-strand (Berg et al 2002).  In the open formation, the two B subunits make what is called the "N-gate" through which the T-segment DNA enters the protein complex. 

 

               C-terminal Domain:  The T-segment DNA exits through this "C-gate" made by the lower portions of the two ParC subunits (Wikipedia).  Initially in the closed formation, the C-gate is opened by the energy that is released when ATP is hydrolyzed a second time (Wang 1996).  The T-segment is then released from the protein through this opened gate formation.

 

 

          Eukaryotes:  Eukaryotic topoisomerase II has all the same essential domains as that of prokaryotes, however it is homodimeric and not heterodimeric (Collins et al 2009).  While prokaryotic topoisomerase II consists of two different dimeric subunits (ParC and ParE), eukaryotic topoisomerase II is made of only one dimer subunit.  Figure 8 shows the generalized structure of topoisomerase II in eukaryotes. 

                                   Figure 8.                  Figure 9.

 

A more complex structure of eukaryotic topoisomerase II is illustrated in Figure 9.  This figure shows the presence of the same key domains in eukaryotic topoisomerase II that were discussed in prokaryotic topoisomerase II.  The CAP domain is shown in cyan, the C-terminal domain in purple, and the homodimer subunits in green and grey.  The DNA is shown in orange.

 

 

 

Topoisomerase IIB - The structure of topoisomerase IIB is similar to that of prokaryotic topoisomerase IIA.  It is a heterodimer containing two different subunits (Wikipedia).  Like prokaryotic topoisomerase II, one subunit dimer possesses the CAP domain and the Toprim domain.  This subunit is referred to as topo VI-A (Wikipedia).  The other subunit possesses the ATPase domain, a H2TH domain, and a transducer domain.  This gene is referred to as topo VI-B (Wikipedia).  Figure 10 shows the homodimeric structure of topo VI.

 Figure 10.

 

 


 

 

IV. Mechanism:

 

     The mechanism by which topoisomerase II functions is called the "two-gate" mechanism (Williams et al 1999).  It binds one strand of DNA, cleaves it, passes another strand through it, and then seals the two cleaved ends back together (Everything2.com).  This is illustrated in Figure 11 and Figure 12.

   Figure 11.            Figure 12.

 

     The two-gate mechanism is so named because of the two "gates" that are opened and closed during topoisomerase II function (Deweese et al 2009).  These gates are the N-gate and the C-gate.  Figure 5 in the previous section shows the location and function of these gates.  The N-gate of topoisomerase IV, named for its location at the N-terminus, is formed by the two ParE subunits (Wikipedia).  In the inactive state, this N-gate is open.  DNA is able to enter the hollow protein core through this open N-gate.  When ATP is hydrolyzed by the ATPase N-terminal domains, the ParE subunits dimerize (Berg et al 2002).  This dimerization changes the conformation of the protein and closes the N-gate, "capturing" the T-segment DNA (Berg et al 2002).  The G-segment DNA is already bound to the DNA-binding core before the T-segment enters the protein.  This ATP hydrolysis leads to the creation and release of ADP and inorganic phosphate.  This inorganic phosphate forms a covalent phosphotyrosine bond between the catalytic tyrosines on the CAP domain and the 5' end of the bound G-segment, breaking the phosphodiester backbone of the DNA (Berger et al 1998).  The G-segment is now cleaved and its ends are secured at the tyrosine active sites, forming a DNA-binding gate (Osheroff et al 1991).  Although it is called a "gate", the DNA-binding gate has nothing to do with the two-gate mechanism name.  The secured ends of the cleaved DNA are not allowed to rotate.  The hydrolysis of ATP at the ATPase domains and the formation of the phosphotyrosine bonds between the DNA and the catalytic tyrosines allows the closing of the N-gate and the cleavage of the G-segment to occur simultaneously (Wang 1996). 

     The T-segment can now pass through the open DNA-binding gate.  Once it has done so, a second round of ATP hydrolysis changes the conformation of the protein again, closing the DNA-binding gate (Holden).  This brings the G-segment ends back together while opening the C-gate at the same time.  The T-segment is then released and the products of the second ATP hydrolysis reset the protein to its original conformation (Berger et al 1998).  Figure 13 summarizes the two-gate mechanism of topoisomerase IV.  The ATPase domains are shown in yellow, the ParE subunits are in red and marked "B" and the ParC subunits are blue and marked "A".

 

   

           Figure 13. 

 


 

 

V. Inhibition:

 

     There are two types of molecules that stunt the function of topoisomerase II: inhibitors and poisons (Fortune et al 2000).

 

          Inhibitors - These molecules reduce and/or terminate topoisomerase II activity by inhibiting the activity of the ATPase domains (Wikipedia).  Through allosteric inhibition, inhibitors can bind to the ATPase domains and change the conformation of the active sites to make them inactive with ATP.  Some examples of inhibitors include HU-331, ICRF-187, ICRF-193, and mitindomide (Wikipedia).

 

          Poisons - In contrast with inhibitors, poisons target the complexes that form between DNA and topoisomerase II during the topoisomerase II process.  One way poisons attack this complex is by increasing the cleavage of the G-segment (Fortune et al 2000).  Another method of attack is preventing the religation of the G-segment ends back together once the T-segment has passed through it (Fortune et al 2000).  Etoposide is an example of a poison that utilizes this method to reduce topoisomerase II activity.  Other poisons include novobiocin, teniposide, doxorubicin, ellipticine, and mitoxantrone.  Figure 14 shows the chemical structure of novobiocin and Figure 15 shows the chemical structure of teniposide.

   Figure 14.                    Figure 15.

 

 

 


 

 

VI. Medical Applications:

 

     During cell division, topoisomerase II works to untangle DNA that is wrapped tightly in chromosomes so that they can be transcribed and/or replicated.  Without topoisomerase II, a cell cannot perform transcription nor replication of its DNA.  In order for a cell to divide and pass on its genes to the daughter cells, it must be able to replicate its DNA.  If a cell cannot do this, then it will not produce successful progeny.  More importantly, if a cell cannot carry out transcription of its DNA, then proteins that are necessary for the cell's survival have no means of being made and the cell will die (Burden et al 1998).  Essentially, preventing topoisomerase II function in a cell is fatal to that cell (Wikipedia).  These truths have had far-reaching effects on the medical field in recent years and have become the basis for much research.  In order to rid the human body of harmful, invasive cells, all one must do is prevent the function of topoisomerase II in those cells. 

 

     Antibiotic Treatments - In recent years, researchers have discovered and further explored the benefits of topoisomerase II inhibitors and poisons in fighting bacterial infections (Biochemistry 3107 Course).  Their findings have advanced and continue to advance the pharmaceutical field in numerous avenues of therapy and treatments. 

          A drug class called the quinolones were introduced to the pharmaceutical world in the 1970's.  Its highly toxic antibiotic qualities were used to treat serious, life-threatening bacterial infections (Oliphant et al 2002).  Figure 16 shows the basic chemical structure of the quinolone family.  Nalidixic acid was the basis for the first generation of quinolones (Wikipedia).  Nalidixic acid was found to block the cleaving and religation mechanism in prokaryotic topoisomerase II (Biochemistry 3107 Course).  The structure of nalidixic acid is shown in Figure 17.  Fluoroquinolones are a subset of the quinolone family and are widely used in clinical treatments today.  Since they are a poison, this class of antibiotics work to interupt DNA replication by attacking the DNA-topoisomerase II complex (Oliphant et al 2002).  Quinolones and fluoroquinolones function to prevent the cleaved ends of the G-segment from dissociating from the tyrosines on the DNA-binding core and religating themselves together (Oliphant et al 2002).  Thus topoisomerase II proteins in target cells are rendered useless causing these cells to soon die.  Ciprofloxacin, a commonly used antibiotic, is among this class of drugs.  Its chemical structure is shown in Figure 18.

Figure 16.             Figure 17.            Figure 18.

 

 

 

 

 

     Anti-cancer Treatments - Topoisomerase II inhibitors and poisons have shown to be useful in treating a variety of cancers in addition to their antibiotic qualities.  Many types of chemotherapy use these agents to control the growth and spread of cancerous cells in the body if not completely irradicating them (Burden et al 1998).  Characteristic of poisons, these anti-cancer agents attack the DNA-topoisomerase II complex by increasing levels of the transient intermediate of the enzymatic topoisomerase II process that is characterized by a cleaved G-segment (Fortune et al 2000).  This intermediate complex is referred to as the "cleavage complex" (Baldwin et al 2005).  The structure of the cleavage complex is illustrated in step 4 of Figure 13 in the "Mechanism" section.  Increasing the level of this cleavage complex intermediate can be done in two ways:  Either increasing the forward reaction rate of cleaving the G-segment or inhibiting the religation of the cleaved G-segment ends (Fortune et al 2000). 

          Etoposide is one of the most commonly used anti-cancer agents in the medical field today.  It can be used to treat a wide range of cancers (Baldwin et al 2005).  It inhibits the religation of the cleaved G-segment DNA strands by stabilizing the cleaved G-segment intermediate (shown in step 4 of Figure 13) (Baldwin et al 2005).  The chemical structure of etoposide is shown in Figure 19. 

          Doxorubicin ultimately works toward the same goal as etoposide, inhibiting the religation of the cleaved G-segment.  The mechanism by which doxorubicin does this is not completely understood, but it is believed that it intercalates itself between the nucleotides in the DNA (Wikipedia).  This phenomena is illustrated in Figure 22.  Figure 21 shows the chemical structure of doxorubicin.

          Genistein has anti-cancer properties that work in the opposite way as etoposide and doxorubicin.  Instead of inhibiting religation, it increases the rate of the forward reaction toward the cleaved G-segment intermediate (National Cancer Institute).  It belongs to a group called isoflavones.  Genistein's chemical structure is shown in Figure 20.

Figure 19.      Figure 20.           

 

 

 

Figure 21.               Figure 22.

 

 

 

Comments (1)

Christopher Korey said

at 10:03 pm on Apr 6, 2009

Looks good. When translating it into the page, make sure to provide concise paragraphs that then link out to other, more indepth material. The links should provide the more detailed information that people could follow up. Try to divide the sections by inserting a horizontal bar to make clearMake sure to reference like any other paper--use a similar reference style and then make them active links that use the URLs that you have copied in the reference section. I like the section you have proposed on anti-cancer drugs.

You don't have permission to comment on this page.