Abstract
The experimental construction of a double-stranded DNA microcircle of only 42 base pairs entailed a great deal of ingenuity and hard work. However, figuring out the three-dimensional structures of intermediates and the final product can be particularly baffling. Using a combination of model building and unrestrained molecular dynamics simulations in explicit solvent we have characterized the different DNA structures involved along the process. Our 3D models of the single-stranded DNA molecules provide atomic insight into the recognition event that must take place for the DNA bases in the cohesive tail of the hairpin to pair with their complementary bases in the single-stranded loops of the dumbbell. We propose that a kissing loop involving six base pairs makes up the core of the nascent dsDNA microcircle. We also suggest a feasible pathway for the hybridization of the remaining complementary bases and characterize the final covalently closed dsDNA microcircle as possessing two well-defined U-turns. Additional models of the pre-ligation complex of T4 DNA ligase with the DNA dumbbell and the post-ligation pre-release complex involving the same enzyme and the covalently closed DNA microcircle are shown to be compatible with enzyme recognition and gap ligation.
Highlights
DNA circularization results from the covalent bonding of the two ends of the same linear DNA molecule
The combination of molecular modeling and uMD simulations has allowed us to characterize the different DNA structures involved in the construction of the smallest, to the best of our knowledge, double-stranded DNA (dsDNA) microcircle reported to date [17]
Our 3D models of the single-stranded D42 dumbbell and the P42 hairpin constructs provide atomic insight into the recognition event that must take place for the DNA bases in the cohesive tail of the hairpin to pair with their complementary bases in one of the single-stranded loops of the dumbbell
Summary
DNA circularization results from the covalent bonding of the two ends of the same linear DNA molecule. The formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in double-stranded DNA (dsDNA) is catalyzed by DNA ligases. Depending on the DNA molecule undergoing cyclization, single-stranded DNA (ssDNA) and dsDNA circles can be obtained. Different sizes of circular DNAs are naturally found in viruses, as well as in prokaryotic and eukaryotic cells, where they carry out multiple functions. The single chromosome that is typically present in bacteria and the extrachromosomal plasmids that are used to transfer genetic information from one individual to another are circular dsDNA molecules. Circular dsDNA can be found in plant chloroplasts and animal mitochondria [2]
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