Dynamics of spontaneous flipping of a mismatched base in DNA duplex

Abstract

DNA base flipping is a fundamental theme in DNA biophysics. The dynamics for a B-DNA base to spontaneously flip out of the double helix has significant implications in various DNA-protein interactions but are still poorly understood. The spontaneous base-flipping rate obtained previously via the imino proton exchange assay is most likely the rate of base wobbling instead of flipping. Using the diffusion-decelerated fluorescence correlation spectroscopy together with molecular dynamics simulations, we show that a base of a single mismatched base pair (T-G, T-T, or T-C) in a doublestranded DNA can spontaneously flip out of the DNA duplex. The extrahelical lifetimes are on the order of 10 ms, whereas the intrahelical lifetimes range from 0.3 to 20 s depending on the stability of the base pairs. These findings provide detailed understanding on the dynamics of DNA base flipping and lay down foundation to fully understand how exactly the repair proteins search and locate the target mismatched base among a vast excess of matched DNA bases.fluctuation spectroscopy | integrated tempering sampling | rate constants | free-energy landscape A base in normal B-DNA spontaneously swinging out of the double helix to an extrahelical position is known as spontaneous base flipping. The dynamics of such base flipping is a fundamental issue in DNA biophysics. It is also related to how DNA repair or modification proteins search and fix the lesion bases to maintain the genome integrity or modify the DNA. Although extensive structural studies have found that many DNA base repair/modification proteins completely flip their target base out extrahelically (so-called enzymatic base flipping) (1-5), it is still under debate (6-11) whether the base flipping occurs spontaneously (9, 10, 12) or not (6-8). Accurate information on the dynamics of spontaneous base flipping is therefore of high interest and importance.However, the study of spontaneous base flipping is deemed to be difficult. The probability is extremely low for a single base to flip out of the DNA double helix in the absence of proteins. Hence only sensitive relaxation methods are able to detect such kind of fluctuation under equilibrium. As a well-known relaxation method, NMR has been applied to tackle this problem through the imino proton exchange assay (9,(13)(14)(15)(16)(17). In this assay, it is assumed that the exchange of the imino proton (in either G or T base) with the catalysts in the solution occurs only when the base flips out (13), and the extrapolated imino proton exchange rate at an infinite catalyst concentration is taken to be the base-flipping rate (14,15,17). According to these NMR studies the lifetime of the extrahelical state is on the order of microseconds, and that of the intrahelical state ranges from milliseconds to hundreds of milliseconds, depending on the stability of individual base pairs. MacKerell and coworkers as well as others have done extensive theoretical investigations and found that the target imino proton on the base already becomes accessible...

Base Flipping within the α-Hemolysin Latch Allows Single-Molecule Identification of Mismatches in DNA

“…The data presented here suggest that intrahelical base pair lifetimes deviate significantly from those measured in solution from single molecule florescence. 9 We speculate that the confined context of the αHL latch constriction in which the mismatched base pair is situated leads to shortened intrahelical lifetimes. The decreased lifetime may result from favourable interactions with the lysine side chains, a feature that will also exist in PCNA.…”

“…The extrahelical lifetimes for mismatched base pairs are reported to be in the 10 - 30 millisecond range at 25 °C, 9,48 thus, the detection of such DNA dynamics with αHL is completely plausible. Conversely, reported extrahelical lifetimes for base-flipping at Watson-Crick pairs is on the nanosecond timescale, 5,53 beyond the current capabilities of ion channel recordings, and explains the absence of current modulation for the fully complementary duplex in our experiments.…”

“…5,9 The mechanisms of base-flipping can be passive, 49 where a protein merely identifies an extrahelical base, or active, 50 where a protein is involved in causing base flipping, and/or stabilizing the intrahelical state.…”

“…7,8 Indeed, recent measurements of base flipping with single-molecule florescence in the absence of proteins have generated debate as to whether intrahelical lifetimes are much longer than previously thought. 9 In a biological context, many proteins are believe to stabilize the extrahelical state (increase the extrahelical lifetime) as a part of the mechanism for repair or maintenance of a specific base. 10,11 …”

Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

“…36 Base flipping is expected to be significantly more prominent at mismatch sites because of their dynamic structure relative to a fully complementary duplex. 22, 23, 31 …”