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A model for MPR. (A) Initiation. (B) The initiator (ID) and its amplification. (C) The propagation of ID in the first heat/cool cycle. 

A model for MPR. (A) Initiation. (B) The initiator (ID) and its amplification. (C) The propagation of ID in the first heat/cool cycle. 

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Article
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Repetitive DNA is a periodic copolymer with the intrinsic property of exponential propagation to longer repeats. Microgene polymerization reaction (MPR) is a model system in which a short nonrepetitive homo-duplex DNA evolves to multiple repetitive products during heat-cool cycles. The mechanism underlying this process involves staggered annealing...

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... MPR is initiated when two HD combine to generate an ID ( Fig. 1 A) by an intricate mechanism (22). The modeled propagation behavior is not sensitive to the exact mechanism of initiation; for the sake of simplicity, a molecularity of 2 was assumed for each of complementary DNA strands. The equation formulating this simplified process ( Fig. 1 A) is ...
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... MPR is initiated when two HD combine to generate an ID ( Fig. 1 A) by an intricate mechanism (22). The modeled propagation behavior is not sensitive to the exact mechanism of initiation; for the sake of simplicity, a molecularity of 2 was assumed for each of complementary DNA strands. The equation formulating this simplified process ( Fig. 1 A) is ...
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... initiator (ID) composed of A 2 and B 2 can be amplified (23) by the original HD (composing of A 1 and B 1 ) ( Fig. 1 B) according ...
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... discrepancies between the derived model and the experimental distributions are evident but can be resolved by testable explanations (Fig. 4): a), the overlap between the first two product-distributions after initiation of MPR is ex- plained by the preceding amplification ( (23) and Fig. 1 B). The kinetics of this stage that corresponds to early MPR cycles is different than that of the net propagation, depending on the difference between k Ampl and k Pr . Since the product of the first cycle (crosses in Fig. 4) includes also the parallel amplifica- tion stage, it should be excluded from the comparison between the experimental and the modeled MPR propagation distribu- tions. b), the very low intensities of the HD peaks in the exper- imental distributions can be explained by the leaching of EtBr from DNA during electrophoresis to the cathode during run on gel (Fig. 2 B). Shorter DNA species such as HD are more vulnerable to EtBr leaching and consequently to an underes- timate of their real amount. The same reasoning is valid for the shortest repeats in all distributions thereby explaining the disparity at their left hand part. This disparity decreases as the length increases, validating the assertion of the vulner- ability of EtBr leaching. c), the experimental distributions are skewed to longer lengths whereas the model predicts negative skewness. This means that for every (A i , B j ) involved in the extension step (see Eqs. 3 and 10) the generated product is much shorter than the expected maximum ((iþj-1) in Appendix B). This is consistent with lower processivity of the Vent polymerase ...
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... ( Fig. 1 C) After generation of the initial doublet, the number of repeats is envisioned as expanding according ...
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... experimental value of the amplification efficiency E (Fig. 2 C and (22)) exceeds 0.5, Eq. 15, implying (see Eq. 3) an overlap between two reacting molecules mainly at their 3 0 terminal units rather than uniformly along their whole lengths (as in Appendix B). Unwinding of the reacting duplexes by strand displacement and their intrusion into each other, as does Vent polymerase (26), can explain this high E: shorter overlaps would be favored because stereo hindrance rises with deeper intrusions (see Fig. 6 in (15)). As a consequence, the average length of the product mole- cules is larger, resulting in E > 0.5. Moreover, each 4 min cycle at around the T m used here is likely to allow more than a single extension step thus further enlarging the average length. Larger E values would thus be anticipated under longer heat-cool MPR ...
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... MPR is kinetically divided into two stages, initiation (enhanced by amplification) and propagation (Fig. 1). The former is slower because the initiator (ID) is generated through an unstable nucleation complex (22,23). However, whereas in common chain-growth polymerization reactions that are limited by unstable intermediates (free radicals or activated precursors in chemical (24) or biological (27-29) macromol- ecules, respectively), the initiation of MPR (after a transient period) is not rate limiting because ID is stable. The rate- limiting stage here is propagation, the mechanism of which shares common features with step-growth polymerization processes (24,25): each extension step, Eq. 3, occurs between molecules containing any number of repeats, which gradually increases with cycles (Fig. 2). The common step-growth poly- merization processes proceeding by condensation is of 2nd-order kinetics resulting in a linear growth of the average polymer molecular weight (25). The MPR, on the other hand, is an autocatalytic process resulting in an exponential growth of the number of repeats per polymer molecule (Fig. ...
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... initiate the process, a nonrepetitive HD is duplicated head-to-tail (17), thus generating a minimum repetitive unit termed initial doublet (ID) (22) that is prone to the succeed- ing expansion (Fig. 1 A). This rare process putatively involves bridging of two molecules of nonrepetitive HD by a third in a manner allowing the DNA polymerase to skip the inter-template gap (22). The ID can be amplified by the original HD before overall expansion starts ((23) and Fig. 1 B). The expansion includes staggered annealing of repetitive single strands of varied lengths followed by poly- merization that fills in overhangs ((15) and Fig. 1 ...
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... initiate the process, a nonrepetitive HD is duplicated head-to-tail (17), thus generating a minimum repetitive unit termed initial doublet (ID) (22) that is prone to the succeed- ing expansion (Fig. 1 A). This rare process putatively involves bridging of two molecules of nonrepetitive HD by a third in a manner allowing the DNA polymerase to skip the inter-template gap (22). The ID can be amplified by the original HD before overall expansion starts ((23) and Fig. 1 B). The expansion includes staggered annealing of repetitive single strands of varied lengths followed by poly- merization that fills in overhangs ((15) and Fig. 1 ...
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... initiate the process, a nonrepetitive HD is duplicated head-to-tail (17), thus generating a minimum repetitive unit termed initial doublet (ID) (22) that is prone to the succeed- ing expansion (Fig. 1 A). This rare process putatively involves bridging of two molecules of nonrepetitive HD by a third in a manner allowing the DNA polymerase to skip the inter-template gap (22). The ID can be amplified by the original HD before overall expansion starts ((23) and Fig. 1 B). The expansion includes staggered annealing of repetitive single strands of varied lengths followed by poly- merization that fills in overhangs ((15) and Fig. 1 ...

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Citations

... A number of in vitro models is used to simulate repeat expansion in DNA (Tuntiwechapikul and Salazar, 2002;Ogata and Miura, 2000;Ogata and Morino, 2000;Liang et al., 2004). Microgene Polymerization Reaction (MPR) is one of such models (Itsko et al., 2009). During MPR DNA sequence containing at least two head-to-tail tandem repeats lengthens exponentially under thermocycling conditions. ...
... During MPR DNA sequence containing at least two head-to-tail tandem repeats lengthens exponentially under thermocycling conditions. The complex MPR kinetics was dissected and numerically analyzed (Itsko et al., 2009). Here we address the process in analytical way. ...
... Microgene Polymerization Reaction (MPR) is a feasible experimental system to study the process of propagation. The general equation describing initiation of MPR and its propagation was formulated, solved numerically and tested experimentally in (Itsko et al., 2009). Here, we will make an attempt to conduct analytical analysis for simplified form of the equation to reveal the nature of chain length distribution. ...
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DNA molecules containing repetitive motifs are prone to expand in their lengths. Once there appear a head to tail tandem of two identical DNA sequences in the system, they can propagate indefinitely by the mechanism involving cycles of staggered annealing of complementary DNA strands of variable lengths and polymerase mediated filling-in of the generated overhangs. Microgene Polymerization Reaction (MPR) is an experimental model for expansion of short repetitive DNA to longer lengths. The testable kinetic model of (MPR) was formulated and solved numerically by Itsko et al. in Kinetics of Repeat propagation in the Microgene Polymerization Reaction (2009). Here, the simple cases of MPR were solved analytically. It was found that the repeats propagate according to Gumbel probability density function when the distribution of lengths of obtained polymers follows inverted Gumbel probability density function.
... Intriguingly, the degree of accuracy in repeat sequence is lower in the last copy in both amino acids and nucleotides. The latter may result of an in vivo mechanism that mimics the in vitro microgene polymerization reaction [Itsko et al., 2009 [Itsko et al., , 2011. The SDS-PAGE-observed molecular mass (110 kDa; not shown) of the full-length Cry11Bb2 is higher by ca. ...
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A new gene, cry11Bb2 from a field isolate of Bacillus thuringiensis, was cloned for expression in Escherichia coli. The encoded protein, with a deduced molecular mass of 89.5 kDa, exhibits 97 and 79% identities with the overlap regions of Cry11Bb1 from B. thuringiensis ssp. medellin and Cry11Ba1 from ssp. jegathesan, respectively. It is however longer than Cry11Bb1 by 42 amino acids in its carboxy-terminus, of which 32 comprise 2 tandem repeats additional to the 5 existing in the latter polypeptide. Possible roles for this recurrent motif among Cry toxins and their accessory proteins, and for their encoding genes are proposed.
Chapter
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Background / Purpose: Repetitive DNA is a periodic copolymer with the intrinsic property of exponential propagation to longer repeats. Microgene polymerization reaction (MPR) is a model system in which a short nonrepetitive homo-duplex (HD) DNA evolves to multiple repetitive products during heat-cool cycles. The mechanism underlying this process involves staggered annealing of complementary DNA strands of variable lengths and polymerase-mediated filling-in of the generated overhangs. MPR is considered as a process sharing common features with two polymerization types, chain-growth and step-growth, and significant distinctions from both types were highlighted. The involved reaction stages were formulated and a kinetic model was derived and tested experimentally. The model quantitatively explains MPR propagation. Main conclusion: The MPR was dissected to sub-reactions and their thermodynamics and kinetics were analyzed. Different propensities of various HDs to expand into multiple repeating units were justified in terms of different stabilities of nucleation complexs (NC) engaged in MPR initiation. The proposed models, thermodynamic for initiation and kinetic for propagation, agree satisfactorily with experimental results. The MPR with non-repetitive HD presents an optional chemical evolutionary system in which the thermodynamic advantage of very weak interactions results in biased proliferation of a certain reaction product. The learned approach to optimize MPR is necessary in protein engineering. The importance of studying this phenomenon lies far beyond applied interest; it may reflect primordial molecular evolution of primitive DNA sequences into complex genomes.