This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Molecular nanomachinery utilized in biological systems involves interplay between different molecular components performing specific functions. The interactions between these components and their internal conformational preferences are both dynamic, and can also be sensitive to atomic level changes in individual nucleotides or amino acids. For each macromolecule, the gap between visualizing its structure and understanding its functional properties needs to be traversed by a description of the conformational free energy landscape governing its activity. Molecular dynamics (MD) simulations describing these nanomachines at atomic level of detail are perfectly suited to tackle problems of molecular recognition involving individual components. They will be used in two separate projects: (1) Recognition of siRNA by the RNAi machinery: Specific atomic level characteristics of siRNA (like phosphorylation at the 5'-end, symmetric 3'-dinucleotide overhangs, and a narrow end-to-end length range) are selectively recognized by the RNAi machinery. The previously determined crystal structures do not completely explain the selectivity, MD simulations will be used to probe the dynamics of association and dissociation of siRNA from the RNAi component PAZ domains. The results will be compared to previous structural and thermodynamic data involving such RNA-protein interactions. (2) Mechanics of DNA strand Propagation in Y-Family DNA polymerases: Y-family DNA polymerases allow replication to continue past lesions in the template strand thereby allowing cells to tolerate DNA damage. They have a propensity to skip over template bases to create disastrous single-base deletion mutations. Recent crystal structures determined in my collaborators (Dr. Janice Pata) laboratory have trapped novel base flipped intermediate states involved in strand propagation. The detailed mechanics of the strand propagation by these polymerases will now be studied using potential of mean force calculations determining the free energy landscape connecting these base flipped states to the stacked states of the extending DNA duplex to quantify their relative stability. I am a new principal investigator initiating these projects in my lab, however, I have extensive experience both as a graduate student (with Dr. Alexander MacKerell, Jr.), and as a postdoc (with Dr. Benoit Roux), in developing and applying the tools of molecular dynamics tools to study biomacromolecules, especially involving nucleic acid-protein interaction.
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