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Dynamics of Spontaneous Flipping of a Mismatched Base in DNA Duplex

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Dynamics of Spontaneous Flipping of a Mismatched Base in DNA Duplex Dynamics of spontaneous flipping of a mismatched base in DNA duplex Yandong Yina,b,c,d,1, Lijiang Yanga,c,e,1, Guanqun Zhengf, Chan Gua,c,e, Chengqi Yig, Chuan Hed,f,g, Yi Qin Gaoa,c,e,2, and Xin Sheng Zhaoa,b,c,d,2 aBeijing National Laboratory for Molecular Sciences, bState Key Laboratory for Structural Chemistry of Unstable and Stable Species, cBiodynamic Optical Imaging Center, dDepartment of Chemical Biology and eInstitute of Theoretical and Computational Chemistry, College of Chemistry and Molecular Engineering, and gSynthetic and Functional Biomolecules Center, Peking University, Beijing 100871, China; and fDepartment of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637 Edited* by Rudolph A. Marcus, California Institute of Technology, Pasadena, CA, and approved April 21, 2014 (received for review January 13, 2014) DNA base flipping is a fundamental theme in DNA biophysics. The rate constants measured from imino proton exchange may not dynamics for a B-DNA base to spontaneously flip out of the double necessarily be appropriate for the interpretation of enzymatic helix has significant implications in various DNA–protein interac- flipping studies” (21). In other words, the fluctuation probed by tions but are still poorly understood. The spontaneous base-flip- the NMR studies should be reassigned to base wobbling instead ping rate obtained previously via the imino proton exchange assay of flipping. Another well-known relaxation method, fluorescence is most likely the rate of base wobbling instead of flipping. Using correlation spectroscopy (FCS) coupled with fluorescence reso- the diffusion-decelerated fluorescence correlation spectroscopy to- nance energy transfer (FRET), has been applied to investigate gether with molecular dynamics simulations, we show that a base many DNA conformational dynamics [e.g., DNA bubble breathing of a single mismatched base pair (T–G, T–T, or T–C) in a double- (22) and base zipping–unzipping (23)]. However, FRET measure- stranded DNA can spontaneously flip out of the DNA duplex. The ment is sensitive to distance roughly from 2 to 7 nm, whereas the extrahelical lifetimes are on the order of 10 ms, whereas the intra- single-base flipping takes place at a distance shorter than 2 nm. helical lifetimes range from 0.3 to 20 s depending on the stability of The FRET–FCS study on spontaneous base flipping is therefore the base pairs. These findings provide detailed understanding on the extremely difficult if not impossible. Indeed, we have not been able dynamics of DNA base flipping and lay down foundation to fully to find such kind of reports in the literature. understand how exactly the repair proteins search and locate the On the other hand, many efforts have been made, by fast target mismatched base among a vast excess of matched DNA bases. mixing the double-stranded DNA (dsDNA) with repair proteins, to monitor the enzymatic base flipping (24–26). However, this fluctuation spectroscopy | integrated tempering sampling | rate constants | ensemble fast-mixing strategy cannot provide independent and free-energy landscape direct kinetic information for the spontaneous base flipping. The observed kinetics of the base flipping is the result of coupling base in normal B-DNA spontaneously swinging out of the between protein and DNA, and the derivation of the base-flipping Adouble helix to an extrahelical position is known as spontaneous rate strongly relies on the assumed damage searching model (9, 25). base flipping. The dynamics of such base flipping is a fundamental Nevertheless, an extrahelical base-trapping strategy similar to the issue in DNA biophysics. It is also related to how DNA repair or enzymatic base flipping was adopted previously to measure the out- modification proteins search and fix the lesion bases to maintain the flipping rate of matched base pairs (27), where β-cyclodextrin was genome integrity or modify the DNA. Although extensive structural used as a host molecule to bind and stabilize the extrahelical bases. studies have found that many DNA base repair/modification proteins completely flip their target base out extrahelically (so-called enzy- Significance matic 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 Spontaneous flipping of a single DNA base to an extrahelical information on the dynamics of spontaneous base flipping is there- position is a fundamental issue in DNA biophysics. The dy- fore of high interest and importance. namics of such spontaneous flipping of a lesion or mismatched However, the study of spontaneous base flipping is deemed to base is an important biophysical feature of DNA to discriminate be difficult. The probability is extremely low for a single base to damaged base from normal Watson–Crick paired ones. It is flip out of the DNA double helix in the absence of proteins. therefore closely related to repair enzymes’ function to rec- Hence only sensitive relaxation methods are able to detect such ognize base lesions, a mechanism on which almost all organ- kind of fluctuation under equilibrium. As a well-known relaxation isms rely to maintain their genome integrity. However, it has method, NMR has been applied to tackle this problem through remained challenging to obtain reliable thermodynamics and ki- the imino proton exchange assay (9, 13–17). In this assay, it is netics data on this process. By developing experimental and the- assumed that the exchange of the imino proton (in either G or T oretical techniques, we determined the free energy and rates of base) with the catalysts in the solution occurs only when the base DNA spontaneous base flipping, providing further insights into flips out (13), and the extrapolated imino proton exchange rate at its molecular mechanism. an infinite catalyst concentration is taken to be the base-flipping rate (14, 15, 17). According to these NMR studies the lifetime of Author contributions: Y.Y., L.Y., C.H., Y.Q.G., and X.S.Z. designed research; Y.Y., L.Y., and the extrahelical state is on the order of microseconds, and that C.G. performed research; G.Z., C.Y., and C.H. contributed new reagents/analytic tools; of the intrahelical state ranges from milliseconds to hundreds of Y.Y., L.Y., C.G., Y.Q.G., and X.S.Z. analyzed data; and Y.Y., L.Y., C.H., Y.Q.G., and X.S.Z. milliseconds, depending on the stability of individual base pairs. wrote the paper. MacKerell and coworkers as well as others have done extensive The authors declare no conflict of interest. theoretical investigations and found that the target imino proton *This Direct Submission article had a prearranged editor. on the base already becomes accessible to the solvent for proton 1Y.Y. and L.Y. contributed equally to this work. exchange when the base pair opens to an angle of only 30°, which 2To whom correspondence may be addressed. E-mail: [email protected] or zhaoxs@pku. is still within the potential well of the hydrogen bonding in- edu.cn. – “ BIOPHYSICS AND teraction (18 21). Hence, All of the conformations that undergo This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. exchange cannot be considered as base-flipped states” and “the 1073/pnas.1400667111/-/DCSupplemental. COMPUTATIONAL BIOLOGY www.pnas.org/cgi/doi/10.1073/pnas.1400667111 PNAS | June 3, 2014 | vol. 111 | no. 22 | 8043–8048 Downloaded by guest on September 28, 2021 The formation of the host–guest complex induces melting of DNA bubble breath (22). The five G bases line up on the same strand duplex below its normal melting temperature, and this melting rate of the DNA duplex opposite the TMR-labeled DNA strand. is taken to be the rate of the spontaneous base flipping of matched TMR was labeled to the C base neighboring the G–T mismatch base pairs (27). However, flipping out a single base in a stable to form a PET pair (Fig. S1 and Table S1). We would expect that dsDNA is physically different from opening base pairs during the when the tested G base of the G–T mismatch stays in the melting of a dsDNA molecule. What this study truly measured may intrahelical conformation, the five stacked G bases show a high not directly correspond to the rate of single-base outward flipping, quenching efficiency and TMR is in a dark state, whereas when but the accumulated rate of unzipping all of the base pairs in- the mismatched G base flips out of the DNA duplex, TMR turns stead (21, 28). In addition, the introduction of the host–guest into a bright state because the flipped G base moves away from complex stabilizes the base open state, so that the energetics and the chromophore (Fig. 1A). Considering the spatial distribution kinetics (especially the extrahelical lifetime of the flipped-out of the dye-linker group (37, 38), the fluorescence increase does state) must be drastically different from that in a natural con- not occur unless the base flips out to a significant extrahelical dition. Other host molecules, such as bisacrindine (BisA), were position (>90°). The dynamics of the spontaneous base flipping found to stabilize the double-helix structure when they bind to in the mismatched G–T pair would then be encoded in the a mismatch-containing dsDNA molecule (29), but we have not fluorescence fluctuation (Fig. 1B). The static fluorescence ex- seen a report on the dynamics of the single-base flipping using periment confirmed our hypothesis: the fluorescence of the G–T the extrahelical trapping strategy by these host molecules. The mismatch is higher than that of the G–C match (Fig. 1C) due to fundamental dynamic properties of the spontaneous flipping of the higher probability of the G base to spontaneously flip out in a single base are still missing.
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