Friday, March 30, 2012

Topoisomerase IB.

            The DNA double helix is a good idea. The important nucleic acid information is cloistered inside the helix, a step away from the solvent and other sources of damage. Mutations, lesions, and mistakes from replication can be detected because correct base pairing is necessary for the helix to form. Helical structure also allows for some useful topological shapes. For example, tightly wound storage nucleosomes can be formed for regions not actively being transcribed and even more condensed chromosomal structures can be formed when cell division is imminent. However, any processes that require access to the information (replication or transcription) also require the unwinding of this helix.
              A human genome contains around 2 meters of 2.4 nm-thick helices. Unwinding these thin, long strands with the replication fork or transcription bubble gives rise to a lot of nasty knots. Imagine trying to separate two strands of thread coiled about each other, held in place at the far end, while only being able to hold the two ends. The tension builds up, super-super coils start forming, and.....your genome doesn't get expressed. No more hemoglobin, glycolytic enzymes, or testosterone. Life as we know it ceases. A Protein of the Year competition is out of the question.

DNA supercoiling.
But, wait..... dUUUM DA DUM: Topoisomerase saves the day!
Topos are a special class of enzymes evolved to prevent this very tragedy.
              There exist a variety of topos for your varied untwisting needs. They accomplish minutely different tasks in minutely different ways. Type I cleave only one strand of dsDNA, reducing the linking number (number of times that one strand crosses the other) by 1 at a time, whereas Type II cleave BOTH strands, requiring ATP and cofactors. They both wind and unwind DNA and reduce the linking number by 2 at a time.
               Type I has further subdivisions of IA and IB. IA relax only negatively coiled superhelices, require a Magnesium ion, and form a transitive covalent intermediate with the 5' phosphoryl group of the transiently broken ssDNA. IB (this is us!) require no Magnesium or stretch of ssDNA to function, and attach to the 3' P. This is what makes this particular topoisomerase so interesting... IBs are sort of the self-reliant oddballs of the group. They don't need no metals. They don't need no ATP.

Let's get to know topo IB's structure.

Structural features of DNA Topoisomerase IB.
Quarterly Reviews of Biophysics (2008), 41 : pp 41-101
            Topo IB has an N terminal region, a core domain (parts I-III), a linker, and a C-terminal domain. Parts I and II of the core domain form a cap over bound DNA. This cap is connected to part III of the core by a hinge on one side of the DNA, and a lip on the other. Core part III and the C-terminal domain, connected by a linker, form the catalytic base where the magic happens. Together, the cap and the catalytic base clamp (PAC-MAN style) with some Hydrogen bonds around double-stranded DNA, encircling it with the nice, warm, fuzzy positive charge of its central cavity, sort of like a hug. There are several known and suspected conformations of the enzyme. It is suspected that the open conformation of the enzyme binds selectively to supercoiled, double-stranded DNA. It clamps around it and assumes the closed formation, which has been crystallized.
              Let's look a little closer at the chemical mechanism of topo IB. It is a simple trans-esterification: nucleophilic attack of an active siteTyr-723 on a phosphodiester bond of one DNA strand, forming a covalent 3' phospho(DNA)-tyrosine(enzyme) intermediate. The knicked 5' end of the strand is stabilized non-covalently in a neighboring area. This stabilized 5' free DNA strand and its complementary strand both rotate to relieve superhelical tension. The duplex rotates on an axis parallel to, but tangential to the circular border of the DNA upstream of the complex. Once some superhelical tension is relieved in this downstream DNA, the the original strand is re-ligated. Re-ligation is essentially the opposite mechanism: the 5' OH of the free DNA strand acts as a nucleophile, attacking the intermediate and releasing the enzymatic Tyr as the leaving group. Several conserved active site residues play key roles in general acid-base capacities. The most interesting part of topo 1B is how the enzyme's structure allows it to tightly control the rate and extent of DNA rotation.
Topoisomerase IB 3'P-DNA intermediate formation mechanism.
              The tension in superhelical DNA is stored free energy that provides the impetus for the unwinding. Topo IB has been termed a "controlled swivelase", for it controls the rate and extent of the unwinding superhelices.
Controlled Rotation Mechanism

Stewart, Lance, Matthew R. Redinbo, Xiayang Qiu, Wim G. J. Hol and James J. Champoux. A Model for the Mechanism of Human Topoisomerase I. 
Vol. 279 no. 5356 pp. 1534-1541
            Multiple rotations occur per re-ligation cycle, and this number is dependent on the amount of supercoiling present. The enzyme senses increased thermodynamic energy when there is more superhelical tension. This energy hinders interactions between the DNA and enzyme more forcefully and decreases activation energy barriers so more rotations can occur before re-ligation.

The effect of torque on re-ligation rates and the free energy profile
"The free energy associated with the angle of rotation between the 5'-OH end of the non-covalently held strand and the tyrosine-3'–DNA adduct in the absence (red curve) and presence (green curve) of torque in the DNA. The effect of the torque is to tilt the landscape, decreasing the barrier height. This increases the escape rate k to k' from the well in which the rate of re-ligation is maximal. This effectively decreases the re-ligation probability per turn."
           The speed of rotation is directly related to the surface area of the protein-DNA interface. Researchers have mutated amino acids in the protein-DNA interface to induce less contact and seen increased rotation velocities. This is presumably due to decreased frictional interference. Interactions between downstream DNA and positive regions of the enzyme also stabilize the conformation necessary for re-ligation: 5'OH in correct orientation with 3'P-Topo intermediate. This is otherwise difficult, for newly freed DNA with significant supercoiling is likely to unwind vigorously and end up interacting elsewhere. The interactions between several amino acids and DNA are shown in the following figure, taken from the paper that first published the crystal structure of the enzyme in its closed conformation with the 22 base pair DNA.

DNA-enzyme interactions

Stewart, Lance, Matthew R. Redinbo, Xiayang Qiu, Wim G. J. Hol and James J. Champoux. A Model for the Mechanism of Human Topoisomerase I. 
Vol. 279 no. 5356 pp. 1534-1541
            The linker region of topo IB also plays a role in the regulation of the rate of re-ligation. It appears that interactions between cleaved DNA and the linker region increase the flexibility of the alpha helical linker and lead to faster re-ligation rates. More interactions will occur between the linker and downstream DNA when it has been sufficiently relaxed and is ready to be re-ligated.
            The structure of the enzyme during its controlled rotation action has eluded researchers, but computational studies have shown that DNA rotation is impossible without some conformational change to the closed enzyme structure. So it is postulated that the enzyme's structure changes somewhat during controlled rotation. It is assumed that different regions of the protein stretch when relieving supercoils of differing directionalities. During positive unwinding, it is speculated that the two lips open slightly to allow for less constrained rotation of downstream dsDNA. During negative unwinding, the hinge region of the protein stretches to accommodate the increased spatial demands of DNA swiveling on the other side of the complex (see figure below).

Topoisomerase IB Mechanisms of unwinding + and - supercoils.

Schoeffler, Allyn J., James M. Berger. DNA Topoisomerases: Harnessing and Constraining Energy to Govern Chromosome Topology. Quarterly Reviews of Biophysics. 41:41-100. 2008.
             DNA information is essential, so topo IB is careful with it. It slows rotation of the free DNA by stabilizing interactions between DNA and the inner cavity and cap region of the enzyme. Interactions between DNA and the linker region influence the rate of re-ligation. Through these mechanisms, topo IB regulates the speed of rotation of the free end of the duplex, and the rate of re-forming the strand. All with no ATP or necessary co-factors.
             The wealth of enzymes with intricate structures, important functions, clever mechanisms, and interesting regulation pathways are all irrelevant if topo stops doing its job. None of these enzymes would even exist without DNA transcription, a process absolutely dependent upon Topo's magical untangling ability.

1. Redinbo, Matthew, James Champaux, and Wim JG Holl. Structural Insights into the Function of Type IB Topoisomerases. Current Opinion in Structural Biology. 9(1): 29-36. 1999.
2. Bugreev, D.V., and G.A. Navinsky. Structure and Mechanism of Action of Type IA
DNA Topoisomerases. Biochemistry (Moscow). 74(13): 1467-1481. 2009.
3. Koster, Daniel A., Vincent Croquette, Cees Dekker, et al. Friction and Torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature. 434:671-674. 2005.
4. Schoeffler, Allyn J., James M. Berger. DNA Topoisomerases: Harnessing and Constraining Energy to Govern Chromosome Topology. Quarterly Reviews of Biophysics. 41:41-100. 2008.
5. Patel, Asmita, Lyudmila Yakovleva, Stuart Shuman, et al.  Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site. Structure. 18:725-733. 2010.
6. Yakovleva, Lyudmila, Shengxi Chen, Sidney M. Hecht, et al.  Chemical and Traditional Mutagenesis of Vaccinia DNA Topoisomerase Provides Insights to Cleavage Site Recognition and Transesterification Chemistry. Journal of Biological Chemistry. 283(23): 16093-16103. 2008.
7. Krogh, Berit Olsen and Stewart Shuman. Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB. Journal of Biological Chemistry. 277: 5711-5714. 2002.
8. Wang, James C. Cellular Roles of DNA Topoisomerases: A molecular perspective. Nature Reviews. 3: 432-446. 2002. 
9. Wereszczynski, Jeff, and Ioan Andricioaei. Free Energy Calculations Reveal Rotating-Ratchet Mechanism for DNA Supercoil Relaxation by Topoisomerase IB and its Inhibition. Biophys J.  99(3): 869–878. 2010. 

Saturday, March 10, 2012

Topoisomerase in the Literature

Need to unwind?

Topoisomerase relieves the tension in DNA strands caused during replication by the helicase's procession along the helix toward a confining binding protein. It does so by cleaving and rejoining one strand of the DNA helix. Cleavage is a trans-esterification reaction in which the phosphodiester bond of DNA is attacked by a tyrosine-723, resulting in the formation of a DNA-(3′-phosphotyrosyl)-enzyme intermediate and the expulsion of the 5′-OH nucleotide and the rest of its strand. During strand rejoining, the 5′-OH end attacks the intermediate to reform a phosphodiester bond and expel the tyrosine.
Berit O Krogh1 and Stewart Shuman. Catalytic Mechanism of Topoisomerase IB. Molecular Cell. 5(6): 1035-1041, 1 June 2000.

DNA can never resist Topo's positive core

Topoisomerase I promote the relaxation of DNA superhelix tension. It does not require any metal co-factors. No ATP is consumed in the reaction. A highly positive core provides an attractive binding pocket for DNA. Two subdomains form a "cap" to the enzyme, which contacts the other sub-domains at only two lips, effectively forming a clamp around DNA.

Electron density map of covalent complex near active site. This shows the 3-phosphate-tyrosine bond between Tyr-723 and the broken strand of DNA.

A) Colored subdomains of the enzyme. B) View of enzyme down pore, with domains colored as in A. Central pore offers a highly positive binding region for DNA. The "cap" of the enzyme contacts core subdomain III at two lips (residues 367-369 of subdomain I and residues 497-499 of subdomain III).

 Matthew R. Redinbo,* Lance Stewart, Peter Kuhn, James J. Champoux, Wim G. J. Holt. Crystal Structures of Human Topoisomerase I in Covalent and Noncovalent Complees with DNA. Science 279 (5356). 6 March, 1998.1504-1513.

Cancer treatments target Topoisomerase 
Topoisomerase IB is overexpressed in mamalian tumor cells because of their increased replication. Camptothecin (CPT) and its derivatives, FDA approved cancer drugs, stabilize the topoisomerase-DNA covalent intermediate, preventing the 5'-OH of the free DNA strand from attacking and re-forming a phosphodiester bond with its partner. During S Phase, the replication forks collide with the topoisomerase-DNA complex and convert the single-strand breakage into a double strand breakage, and eventually apoptosis is signaled. CPT was originally isolated from the bark and stem of the Chinese tree Camptotheca acuminata, and it is presumed that its linear structure stabilizes the intermediate.
CPT- wikipedia
Rotated +1 Nucleoside model for Top1 interaction with dsDNA and 10-OH CPT. CPT is the spherically represented structure. Cyan blue is DNA, and yellow and red indicate enzymatic residues.

Laco, Gary S. Evaluation of Two Models for Human Topoisomerase I Interaction with dsDNA and Camptothecin Derivatives.  Public library of sciences. 6(8). 2011.

Wednesday, February 22, 2012


Surface representation of Toposomerase with DNA bound.
Secondary structure motifs.
DNA splicing activity. Bright green shows break in DNA strand.
Close view of break in DNA.
Interactions between DNA and Topoisomerase.