Molecular Models

Although not always possible or absolutely necessary, we have found it useful in most cases to prepare at least preliminary molecular models of the assemblies under consideration. In this case (Fig. 2), molecular models of the Y-Junction recombination intermediate were constructed in Insight II (Accelrys, San Diego, CA). The DNA builder was used to extend the DNA present in the three-dimensional structure of the MHhaI protein-DNA complex (12) obtained from the Protein Data Bank (13) with PDB ID:1MTH. The arms were linked at the center to form a model of the Y-Junction having an accurate sequence. Assisted model building with energy refinement molecular force field (14) was used to minimize the structure while constraining the DNA and protein struc-

Fig. 1. DNA methyltransferase mechanism of action. Methyltransferases make a nucleophilic attack on C6 of cytosine or 5-fluorocytosine. This breaks the 5-6 double bond in the ring and activates C5 for methyltransfer. After the methyl group is transferred from S-adenosyl-[L]-methionine (AdoMet) to C5 of cytosine, the hydrogen at C5 and the enzyme nucleophile at C6 are removed by P-elimination. When fluorine is present at C5, this cannot occur because of the strength of the fluorine-carbon bond. Thus, the progress of the reaction is blocked, leaving a covalent linkage between the enzyme and the cytosine ring targeted by the enzyme. In the case of M^coRII, it is the second cytosine in the CCWGG recognition sequence that is attacked, in which W stands for A or T. MHhal attacks the first cytosine in the GCGC sequence, and human Dnmtl (hDnmtl) attacks the cytosine in the CG dinucleotide.

Fig. 1. DNA methyltransferase mechanism of action. Methyltransferases make a nucleophilic attack on C6 of cytosine or 5-fluorocytosine. This breaks the 5-6 double bond in the ring and activates C5 for methyltransfer. After the methyl group is transferred from S-adenosyl-[L]-methionine (AdoMet) to C5 of cytosine, the hydrogen at C5 and the enzyme nucleophile at C6 are removed by P-elimination. When fluorine is present at C5, this cannot occur because of the strength of the fluorine-carbon bond. Thus, the progress of the reaction is blocked, leaving a covalent linkage between the enzyme and the cytosine ring targeted by the enzyme. In the case of M^coRII, it is the second cytosine in the CCWGG recognition sequence that is attacked, in which W stands for A or T. MHhal attacks the first cytosine in the GCGC sequence, and human Dnmtl (hDnmtl) attacks the cytosine in the CG dinucleotide.

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Fig. 2. Assembly of a three-address nucleoprotein array. A schematic of the final assembly process for the machine is depicted in the upper right. The black panel in the lower right depicts molecular models of the predicted intermediates in the assembly of the three-address array once the duplex Y-Junction itself is formed. In the last frame of the molecular model panel, NLS-M^coRII is depicted as an ovoid with a crooked appendage depicting the NLS peptide. (Figure 2 caption continued on next page.)

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Fig. 2. Assembly of a three-address nucleoprotein array. A schematic of the final assembly process for the machine is depicted in the upper right. The black panel in the lower right depicts molecular models of the predicted intermediates in the assembly of the three-address array once the duplex Y-Junction itself is formed. In the last frame of the molecular model panel, NLS-M^coRII is depicted as an ovoid with a crooked appendage depicting the NLS peptide. (Figure 2 caption continued on next page.)

tures in the arms beyond the 10 bp at the center of the junction. The energies of the resulting structures were minimized with respect to molecular mechanics until the RMS force was <0.1 (kcal/mol)/A. Following molecular mechanics, each structure was further minimized with 1000 steps of simple dynamics. We did not attempt to prepare a molecular model of the nuclear localization signal (NLS)-M*EcoRII fusion protein because a crystal structure for M*£coRII was not available. Current information on this protein (15) suggested that a 30-bp arm on the Y-Junction could accommodate the fusion protein in the environment created by the linking of MHhaI proteins to the adjacent arms of the Y-Junction.

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