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. http://www.csun.edu/~ll656883/readings/reading4.pdf |
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 |
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. Wikipedia. |
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).
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.
Sources:
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.
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.
Sources:
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.