Poster abstracts

Poster number 30 submitted by Besik Kankia

Differential Effects of HIV-1 NC on Strand-exchange Reactions Involving Mismatched DNA Duplexes

Besik Kankia (Department of Chemistry, The Ohio State University), Karin Musier-Forsyth

Abstract:
The HIV-1 nucleocapsid protein (NC) facilitates strand-replacement reactions during reverse transcription. The role of NC in these processes is linked to its nucleic acid chaperone activity, whereby nucleic acids are rearranged into their thermodynamically most stable structure. In the present work, we use an optical quadruplex displacement assay to study the role of NC in strand-replacement reactions. Short DNA duplexes containing two G-A mismatches at different positions were investigated: adjacent mismatches in the middle of the duplex (Reaction 1), single mismatches separated by 11 nucleotides (Reaction 2), and adjacent mismatches at the end of the duplex (Reaction 3). The reactions were studied as a function of temperature and strand concentration, which revealed that strand replacement proceeds through two different pathways: dissociative (monomolecular) and sequential displacement (bimolecular). The former requires complete dissociation of the reactant duplex followed by rapid formation of product duplex, and the latter pathway assumes that the chase strand initially base pairs to the reactant duplex at the most unstable part followed by a fast displacement process. As expected, the activation energy of the sequential displacement pathway is lower than that of the dissociative pathway for Reactions 1 and 2. Surprisingly, in the case of Reaction 3 the opposite is true. We hypothesize that this is due to the formation of a bimolecular intermediate, most likely a three-stranded DNA duplex stabilized by G-quartets. NC accelerates the strand-replacement kinetics by 1-2 orders of magnitude, and lowers the activation energy of the monomolecular pathway by 10-15 kcal/mol for all reactions. NC also lowers the activation energy of the sequential displacement pathway for Reaction 1 and 2 by 25 kcal/mol and 13 kcal/mol, respectively. In the presence of NC, the dissociative pathway completely dominates the kinetics of Reaction 3, which could be explained by more favorable formation of the bimolecular intermediate due to NC’s nucleic acid aggregating ability.

Keywords: DNA strand-exchange, HIV-1 nucleocapsid protein, Kinetics