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The Origin of Long Template Replication.

The Origin of Long Template Replication. Chrisantha Fernando Eörs Szathmáry* Centre for Computational Neuroscience and Robotics. Dept of Informatics. University of Sussex. Brighton, BN1 9QH

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The Origin of Long Template Replication.

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  1. The Origin of Long Template Replication. Chrisantha Fernando Eörs Szathmáry* Centre for Computational Neuroscience and Robotics. Dept of Informatics. University of Sussex. Brighton, BN1 9QH *Dept ofPlant Taxonomy and Ecology, Eötvös University, and Collegium Budapest (Institute for Advanced Study), Szentháromság u. 2, H-1014 Budapest, Hungary

  2. Why is long template replication important? Unlimited heredity.

  3. Above 6 nucleotides in length, templates are stuck tightly by h-bonds. Making covalent (P) bound Separation is the rate-limiting step. H-bonds Product Monomers Template

  4. Ribozymes > 6nt in length are needed to replicate templates > 6nt, but how could these have arisen without long template evolution? • Eigen’s Hypercycles. • Hypothesis: Non-enzymatic long template replication originated in replicating vesicles. • Results: If oligomers can be incorporated into long templates faster than they are produced, then oscillating temperature between 0 and 80oC allows long template replication. This is favored by high [polymer] and increasing p-bond formation rate. • Working conclusion: These favorable conditions are most likely to obtain in replicating vesicles.

  5. Chemical pathways detailed in Ganti (2003) OUP “The Principles of Life”. and Ganti (2004) Kluwer “Chemoton Theory Vol I and II”

  6. A Stochastic Model of Template Dynamics.

  7. Stochastic discrete model with explicit p-bonds and h-bonds represented on a 2D grid (2o struc). • Formation or breakage of a bond depends on the local polymer configuration (and [monomer]). • Association reactions between polymers is also modeled. • One explicit polymer represents 3000 – 300000 real polymers. [Polymer classes are the unit of representation.] • Model can be coupled to a deterministic continuous model of the chemoton metabolism and membrane dynamics.

  8. Polymer Reactions Converting Rates to Probabilities, P(AB) = 1-exp-dt*Rate dt = 10-9 seconds. Simple Euler Integration.

  9. Hydrogen Bond Breakage. • Stacking dependent h-breakage probability. • Rate = Aconfe(Ea-Kn)/RT • Aconf is configuration dependent (see below). • Ea = 31.4kJ • K = 0.01 and n = no. free h-bond sites, apx equal to length of breathing ends. This models effect of increased entropy of breathing ends.

  10. Configuration Dependent Hydrogen Bond Formation. • Occurs at fixed rate 106 sec-1 at the configuration below…

  11. Configuration Dependent Phosphodiester Bond Formation. • Occurs at Rate = S*0.6e-25/RT sec-1 at the configuration below…………………. • S is a scaling factor 1010 and 1011 required for reasonable simulation times. Also applied to p-break rate.

  12. Zipper Mechanism • H-bonds can form between opposite monomers along a double strand, at fixed rate 106 sec-1. • The dark p-bond has 4 possible places where a h-bond could be formed upon it. • If there is a p-bond opposite one of these 4 positions, a h-bond is formed at the rate above.

  13. Novel Monomer Attachment. • Rate =106 [V’] , i.e. catalysed h-bond formation * [V’]. • Monomer attachment is in competition with OTHER polymers • for attachment to potential h-bond sites. 100* rate of attachment if stacked.

  14. Polymer Association. • Association is tested between each possible ordered polymer pair. • Association rate = 106*(n*[polymer unit]), • I.e. proportional to catalysed h-bond formation rate, number of potential h-bond sites per polymer, unit concentration of a single polymer (3000 or 300000/Volume*Avogadro). • If a random number between 0 and 1 is > P(assoc) then polymers are associated randomly in a legitimate secondary structure.

  15. P-bond Degradation. • Rate = S*1.32 * 1012e-110/RT sec-1 for single strands < 14-mer in length. • Longer single strands are assumed to have negligible p-bond breakage due to hairpin formation. • Double strands are assumed to have negligible p-bond breakage. • S is the same scaling factor used to scale P-bond formation rate.

  16. Control Experiments.

  17. Melting Temperature v. Length • 1/Tm = A + B/N. Tm is melting temperature, A & B are experiment dependent constants, and N is strand length.

  18. Tm v. [Polymer]. • 1/Tm = A’ – B’lnC. • C = [Polymer].

  19. Flow Reactor Results. At high temperatures elongation occurs, replication is not observed. At low temperatures oligomers out-compete long strands. High [Polymer]/[Monomer] Temperature = 350K. Low [Polymer]/[Monomer] Temperature = 300K.

  20. Type I mechanism. High [Monomer] Low [Polymer] Low Temp. Produces oligomers which can later be incorporated in to long strands. Type II mechanism High [Polymer] Low [monomer] High Temp. Elongates and replicates long strands by oligomer and monomer incorporation .

  21. Failed attempts at improving the type I mechanism. • Failure to replicate long strands with the type I mechanism alone is surprisingly robust to modification of many aspects of nucleotide kinetics. This is because, whatever you do, short strands always replicate faster than long strands by using the type I mechanism, so always out-compete long strands in the end.

  22. The following interventions failed • Instantaneous p-bond formation failed because incomplete strands were still lost. • Increased monomer stacking rate failed. • Altering breathing rate failed because rate is the same for parent and incomplete novel strands. • A simulated enzyme that preferentially stabilized hydrogen bonds on incomplete strands failed. • A simulated enzyme that preferentially stabilized h-bonds on only the incomplete strands on parent strands greater than 7 nt, did not result in polymers > 7nt.

  23. A Mechanism for Long Template Replication.

  24. The main factors preventing long template replication were… • Competition by successfully unzipping short replicators. • No unzipping of long double strands. • Premature detachment of incomplete copies from longer strands. The model predicts that a solution is to oscillate temperature at high [polymer].

  25. Temperature Oscillation

  26. Replication can still occur at decreased [Polymer] = 0.01M – 0.005M The Type II mechanism occurs at a lower rate and temp oscillation may not be neccesary. Fixed Temp = 350K Temp Oscillation 270K-350K

  27. BUT !!! Reduced p-bond formation rate means that oligomers out compete long strands. Type I mechanism >> Type II mech.

  28. Requirements for Replication by the Type II mechanism. • Incorporation of oligomers by staggered re-association must exceed their rate of production by de novo synthesis by incomplete strand separation and by replication of oligomers. • This requires high p-bond formation rates, high temperatures and high [polymer]. Temperature oscillation is not essential, but allows control of the replication mechanism.

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