J. Phys. Chem. B 2007, 111, 5757-5766
5757
Site-Specific Dynamics of Strands in ss- and dsDNA As Revealed by Time-Domain Fluorescence of 2-Aminopurine T. Ramreddy,† B.J. Rao,*,‡ and G. Krishnamoorthy*,† Department of Chemical Science and Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India ReceiVed: December 21, 2006; In Final Form: March 4, 2007
It is well recognized that structure and dynamics of DNA strands guide proteins toward their cognate sites in DNA. While the dynamics is controlled primarily by the nucleotide sequence, the context of a particular sequence in relation to an open end could also play a significant role. In this work we have used the fluorescent analogue of adenine, 2-aminopurine (2-AP), to extract information on site-specific dynamics of DNA strands associated with 30-70 nucleotides length. Measurement of fluorescence lifetime and anisotropy decay kinetics in various types of DNA strands in which 2-AP was located in specific positions revealed novel insights into the dynamics of strands. We find that in single-stranded (ss) DNA, the extent of motional dynamics of the bases falls off sharply from the very end toward the middle of the strand. In contrast, the flexibility of the backbone decreases more gradually in the same direction. In double-stranded (ds) DNA, the level of basepair fraying increases toward the ends in a graded manner. Surprisingly, the same is countered by the presence of ss-overhangs emanating from dsDNA ends. Moreover, the extent of concerted motion of bases in duplex DNA increased from the end to the middle of the duplex, a result which is both striking and counterintuitive. Most surprisingly, the two complementary strands of a duplex that were unequal in length exhibited differential dynamics: the longer one with overhangs showed a distinctly higher level of flexibility than the recessed shorter strand in the same duplex. All these results, taken together, provoke newer insights in our understanding of how different bases in DNA strands are endowed with specific dynamic properties as a function of their positions. These properties are likely to be used in facilitating specific recognitions of DNA bases by proteins during various DNA-protein interaction systems.
1. Introduction The information content of DNA is often assumed to be solely deposited in its sequence. While this assertion holds in a vast majority of situations, there are striking examples wherein the information retrieval from a sequence is modulated by the position of the sequence with respect to an open end or even by the conformational states of chromatin organization.1 Such extra-sequence information could primarily originate from position dependence of structure and dynamics of DNA stretches. Despite the general realization that dynamics of DNA plays a vital role in DNA-protein interaction, the level of our understanding on nucleic acid dynamics2-7 is rather limited. Furthermore, information on position-dependent dynamics is even more stark by its sparseness.8-13 Position-dependent dynamics is implicated in a variety of situations such as specific and sequence-independent binding by regulatory proteins to dsDNA ends (blunt-ended or single-stranded overhangs),14 ssDNA and junctions of ssDNA/dsDNA.15 On the other hand, another quandary relates to a wide dispersion of highly degenerate transcription factor-binding sequences in genomes that would seemingly result in a large number of nonspecific interactions or even promiscuous bindings to nonfunctional pseudosites.16 The basis of observed binding specificity among such apparently degenerate sites may stem from base-specific dynamics as a function of sequence context. There are also * Authors for correspondence. † Department of Chemical Science. ‡ Department of Biological Sciences.
situations where only a select few sites in highly preponderant small target sequence sites in a DNA strand seem to dictate high binding specificity toward recognition by proteins.17 This again may have its origins in hitherto unexplored positiondependent base-pair dynamics in DNA strands. Fraying of end segments of dsDNA has been inferred in several situations18 and implicated in specific binding of many DNA binding proteins at the end. Specific recognition of dsDNA ends by proteins such as translin19 is likely to be mediated by end-specific DNA dynamics. For example, human DNA repair proteins Ku70 and Ku80 bind specifically to blunt-ended duplexes and initiate nonhomologous end joining pathways. Flexibility of dsDNA is an important factor in double-strand breaks and chromatin remodeling,20,21 formation of DNA minicircles,22 etc. and has been studied by a variety of techniques including cyclization kinetics23 and direct measurement of persistence length at single-molecule level using atomic force microscopy.24 Interpretations that rely solely on strand sequences look untenable, begging for newer explanations encompassing strand dynamic aspects where it has been demonstrated that endonucleases acting on the junctions of single- and doublestranded DNA are highly influenced by the effects of polarity, length, configurations of junctional arms, etc. even within the sets of very similar substrate sequences.25 Site-specific internal dynamics of RNA has been recently studied by NMR spin relaxation methods and is correlated with adaptive recognition of target molecules.7 Similar functionally relevant motional modes are likely to be present in the DNA
10.1021/jp068818f CCC: $37.00 © 2007 American Chemical Society Published on Web 05/01/2007
5758 J. Phys. Chem. B, Vol. 111, No. 20, 2007 world aimed toward directing specific protein targets. Singlestranded DNA overhangs are often generated in DNA transcription, replication, and recombination. Such single-stranded overhangs are involved in specific binding of several proteins.26 RecA in Escherichia coli and Rad51 in higher organisms bind to such tailed duplexes and initiate repair processes via homologous recombination. Tailed dsDNA substrates permit Rad51 protein to promote DNA strand invasion of both 3′- and 5′-ends with similar efficiencies. It is also known that human Rad52 preferentially binds to the 5′-end of ssDNA over the 3′end.15 Thus, it is very important to study the dynamics of ssDNA as well as that of dsDNA with single-stranded overhangs. All the above-cited examples underscore the dire need for new understanding about the DNA base dynamics in relation to strand polarity, neighbor sequence contexts, distance from DNA free ends, secondary structural forms of DNA (single-, double-, multiple-stranded forms, and the junctions that connect them), etc. in order to fully relate the structures to the observed functional specificities. In this work, we have used the fluorescent analogue of adenine, 2-aminopurine (2-AP)27-30 to monitor the position dependence of the dynamics of base and backbone of ss- and dsDNA. Repeats of adenine rather than mixed sequences were used in this work to ensure that the results will reflect the effect of the position on the sequence rather than the identity of the bases on either side of 2-AP; therefore, any effects that are likely to arise from sequence contexts were obviated in the chosen design. 2-AP, which forms a base-pair with thymine, has been very effectively used to study both specific and nonspecific aspects of structure and dynamics of DNA in a wide variety of situations.31-38 2-AP can be selectively excited in the presence of DNA, RNA, and proteins, since its absorption occurs at wavelengths longer than those of the nucleic acid bases and aromatic amino acids. Fluorescence of 2-AP is strongly quenched when incorporated in DNA, and the level of quenching is sensitive to local and global changes in DNA conformation.39-41 Furthermore, fluorescence intensity of 2-AP is quite sensitive to stacking interactions with neighboring bases as well as on the dynamics of near neighbors. Free 2-AP in solution has a fluorescence quantum yield of 0.68 at pH 7,42 and it has a single fluorescence decay lifetime of 11.6 ns. Incorporation of 2-AP into an oligonucleotide or oligodeoxynucleotide duplex leads to heterogeneous fluorescence decays with time constants ranging from 95%) single strands were converted to duplexes as ascertained by the autoradiographic analyses of an aliquot of the same on 10% native polyacrylamide gel to which trace amounts of 5′-32P-labeled ssDNA strand was added to the mixture prior to annealing, as described earlier.49-51 In general, samples contained 20 µM of nucleotide concentration of DNA bases in 20 mM (pH 7.5) Tris buffer, 10 mM magnesium acetate, and 1 mM EDTA for all the fluorescence measurements. Measurements were done at room temperature 24((1)°C unless mentioned otherwise. 2.3. Fluorescence Measurements. Steady-state fluorescence excitation spectra were carried out on a SPEX Fluorolog FL111 T-format spectrofluorimeter. The excitation source was a Xe lamp. All the fluorescence spectra were corrected for the spectral sensitivity of the photomultiplier (Hamamatsu R928A). The bandwidth used was between 1 and 2 nm. Appropriate filters were used before the emission monochromator to avoid the excitation light entering it. Time-resolved fluorescence intensity and anisotropy decay of 2-AP was measured by employing CW-passively modelocked frequency-doubled Nd:YAG laser (Vanguard, Spectra Physics, U.S.A.)-driven rhodamine 6G dye laser which generates pulses of width ∼1 ps. 2-AP in DNA was excited by using the second harmonic output (310 nm) of an angle-tuned KDP crystal. Fluorescence decay curves were obtained by using a time-correlated single-photon counting setup, coupled to a microchannel plate photomultiplier (model 2809u; Hamamatsu Corp.). The instrument response function (IRF) was obtained at 310 nm using a dilute colloidal suspension of dried nondairy coffee whitener. The half width of the IRF was ∼40 ps. Time per channel was 39.2 ps. The samples were excited at 310 nm, and the fluorescence emission was collected through a 310 nm cutoff filter followed by a monochromator at 370 nm with a collection bandwidth of 10 nm. The cutoff filter was used to prevent scattering of the excitation beam from the samples. The number of counts in the peak channel was at least 10 000. In fluorescence lifetime measurements, the emission was monitored at the magic angle (54.7°) to eliminate the contribution from the decay of anisotropy. In time-resolved anisotropy measurements, the emission was collected at directions parallel (I|) and perpendicular (I⊥)) to the polarization of the excitation beam.
Dynamics of Strands in ssDNA and dsDNA
J. Phys. Chem. B, Vol. 111, No. 20, 2007 5759
Figure 1. DNA constructs used in this work.
The anisotropy was calculated
r(t) )
I|(t) - I⊥(t)G(λ) I|(t) + 2I⊥(t)G(λ)
Time-resolved anisotropy decays were analyzed based on the model
(1)
where G(λ)) is the geometry factor at the wavelength λ of emission. The G factor of the emission collection optics was determined in separate experiments using a standard sample (Nacetyltryptophanamide) for which the rotational correlation time was 0.1 ns and fluorescence lifetime was 2.9 ns. 2.4. Analysis of Results. Experimentally measured timeresolved fluorescence decay data, F(t), is a convolution of the instrument response function, R(t), and the intensity decay function of the sample, I(t):
F(t) )
∫0t R(s + δ)I(t - s)ds
Ri exp(-t/τi) ∑ i)1
(4)
I⊥(t) ) I(t)[1 - r(t)]/3
(5)
r(t) ) r0{β1 exp(-τ/φ1) + β2 exp(-τ/φ2)}
(3)
where ∑iRi ) 1.0, by the iterative deconvolution method.52 Correction factors for the parameters (Ri and τi) in successive iterations were determined by the application of Marquardt’s method in nonlinear least-squares analysis.53 Numerical calculation of the convolution integrals for intensity and partial derivatives were done using the Grinvald-Steinberg recursion equations.52 The mean lifetime τm ) ∑iRiτi gives us information on the average fluorescence yield of the system.
(6)
where r0 is the initial anisotropy, βi is the amplitude of the ith rotational correlation time φi such that ∑iβi ) 1.The shorter component φ1, representing the internal motion of 2-AP, could be modeled as due to hindered rotation.54 The angular range of this hindered rotation was calculated by the isotropic diffusion inside a cone.54 The semi-angle θ of the cone is given by
θ ) cos-1
(2)
where δ is the shift parameter, which is a fraction of the time per channel. R(t) is experimentally determined. I(t) is a function assumed to describe the fluorescence dynamics of the sample. Decay data analysis involves the determination of the best values for the unknown parameters in I(t). Time-resolved fluorescence decay data were fitted to a function that is a sum of discrete exponentials
I(t) )
I|(t) ) I(t)[1 + 2r(t)]/3
{21 [[(1 + 8(β )
) ] - 1]
1/2 1/2
2
}
(7)
We have obtained, in several samples, lifetimes and rotational correlation times in the range of 0.1-0.3 ns. Since these values are too close to both the width of the instrument response function and the time/channel used, both of which are ∼40 ps, their reliability might seem uncertain. In order to check the reliability of estimating such short time constants, we measured the rotational correlation time (φ) of a small molecule Nacetyltryptophanamide (NATA) in glycerol-water mixtures with viscosity (η) in the range 1-5 cP. The values of Φ estimated were 0.061 ( 0.025, 0.123 ( 0.036, 0.226 ( 0.028, and 0.311 ( 0.027 ns when the solvent viscosities were 1.0, 2.1, 4.4, and 5.4 cP, respectively, as expected from the StokesEinstein relationship (Φ ) ηV/kT). 3. Results Current study involved analyses of three commonly found forms of DNA in cells during genomic events, namely, singlestranded (ssDNA), double-stranded (blunt-ended dsDNA) and
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TABLE 1: Parameters Associated with Fluorescence Intensity Decay and Fluorescence Anisotropy Decay in ssDNA
sample 1
A30(1)
2 3 4
A30(2) A30(4) A30(15)
5 6 7
A30(28) A60(30) A-template
τ1(R1)
fluorescence lifetime, ns (amplitude) τ2(R2)
τ3(R3)
mean lifetime τm (ns)
0.55(0.35) 0.55(0.30)b 0.56(0.24) 0.56(0.25) 0.70(0.24) 0.74(0.20)b 0.70(0.30) 0.59(0.22) 0.61(0.22)
1.66(0.50) 1.63(0.54)b 1.70(0.62) 2.3(0.63) 2.3(0.64) 2.4(0.61)b 2.3(0.57) 2.2(0.65) 2.2(0.65)
4.4(0.15) 4.2(0.16)b 6.0(0.14) 6.3(0.12) 5.8(0.12) 5.3(0.19)b 5.9(0.13) 5.7(0.13) 5.7(0.13)
1.68 1.71b 2.03 2.35 2.34 2.62b 2.29 2.30 2.31
rotational correlation time, ns (amplitude) φ1(β1) φ2(β2) 0.15(0.60) 0.20(0.58)b 0.20(0.44) 0.15(0.43) 0.14(0.40) 0.20(0.38)b 0.18(0.44) 0.17(0.43) 0.14(0.39)
1.1(0.40) 1.2(0.42)b 1.4(0.56) 2.0(0.57) 3.0(0.60) 3.3(0.62)b 1.9(0.56) 4.0(0.57) 2.8(0.61)
a The number in parenthesis refers to the position of 2-AP from the 5′-end. Maximum errors are ∼20% in the values of τ1, R1, and φ1 and ∼10% for other parameters except the case of τm for which the error is
