High Base Pair Opening Rates in Tracts of GC Base Pairs

Utz Dornberger, Mikael Leijon, Hartmut Fritzsche
1999 Journal of Biological Chemistry  
Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1 H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much
more » ... horter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed. Many DNA-binding proteins are highly selective in their recognition of particular DNA sequences. Besides sequencespecific hydrogen bonding and van der Waals interactions, sequence-dependent structure and dynamics of DNA are likely to play an important role in DNA-protein interaction. In addition, the adaptability of a DNA sequence element to structural changes necessary for sequence-specific interaction is important in recognition (1). Base pair opening is required in many fundamental processes in the cell, for example, transcription and recombination. Recently, base pair opening was found to participate in a novel mode of protein-DNA interaction. The crystal structures of the M.HhaI 1 and M.HaeIII cytosine-5-methyltransferases in complex with their DNA recognition sequences showed the target base completely flipped out from the helix (2, 3). Cytosine-5-methyltransferases usually recognize a sequence of four GC base pairs (4). M.HhaI and M.HaeIII recognize 5Ј-GCGC-3Ј and 5Ј-GGCC-3Ј, respectively. A pertinent question is whether these enzymes actively expel the target base from the helix stack or capture a transient spon-taneous opening. It has been shown that M.HhaI binds more tightly when a mismatch is created in the recognition sequence by replacing the target cytosine by any other base or an abasic site but not 5mC (5, 6). The enhanced binding was attributed to the lower energy required for opening a mismatched base pair upon formation of the binary complex. Hence, the base pair dynamics at the cytosine target site seems to contribute to the specificity of the cytosine-5-methyltransferases. These findings prompted us to investigate the base pair dynamics of tracts of GC base pairs. Measurements of base pair dynamics yield information about stability and structure of the double helix. Furthermore, studies of base pair opening in DNA interacting with drugs (7, 8) and hybridized with uncharged PNA (peptide nucleic acids) (9) have provided new clues to the mechanisms of spontaneous helix breathing. Another important finding is that tracts of AT base pairs exhibit anomalously long base pair lifetimes. Although the origin of this effect is uncertain, it seems likely that AT tracts form a particularly stable structure cooperatively, a so-called BЈ-DNA helix (10). Hence, increased base pair lifetimes are indicative of this type of structure. In general AT base pair lifetimes have been found to be in the range 1-5 ms at 15°C, except for AT tracts where lifetimes longer than 100 ms have been observed (11). For GC base pairs, lifetimes about 10 times longer than for AT base pairs usually have been observed (12), as one might expect from the presence of an additional hydrogen bond in the GC base pair. However, most studies have concerned isolated GC base pairs (9, 11, (13) (14) (15) (16) . Representative values for isolated GC base pair lifetimes are compiled in Table I . No study has been undertaken of the base pair opening dynamics in tracts of GC base pairs. Since the binding affinity of M.HhaI increase with the lability of the target base pair (5, 6), one would not expect base pair dynamics to contribute to the specificity of the cytosine-5-methyltransferases in view of the hitherto observed higher stability of GC base pairs. However, in the present study it is shown that tracts of GC base pair have unusually rapid base pair dynamics contrary to isolated GC base pairs and in striking contrast to AT tracts. EXPERIMENTAL PROCEDURES Sample Preparations and Base-catalyst Titrations-All oligonucleotides were either synthesized by using automated phosphoramidite chemistry on a DNA synthesizer (Applied Biosystems model 394) or purchased from Cybergene Inc. (Sweden). The oligonucleotides were purified by reverse-phase high performance liquid chromatography and desalted by Sephadex G-25 column chromatography. The NMR samples were prepared by dissolving the oligonucleotides in a 3 mM borate buffer at pH 8.8 containing 100 mM NaCl (90% H 2 O and 10% D 2 O). The duplex concentrations were in the range 1.1-1.8 mM. Ammonia was added in appropriate amounts from a 6.6 M stock solution at pH 8.8. Two separate titrations were carried out for each duplex and the exchange-time ( ex ) data were combined and linearly fit versus the inverse base-concentration (1/[B]) via Equation 1 (13). ex ϭ op ϩ 1 ␣K d k i ͓B͔ (Eq. . 1 The abbreviations used are: M.HhaI, M.HaeIII, and M.EcoRI, methyltranferases; 5mC, 5-methylcytosine; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PNA, peptide nucleic acid.
doi:10.1074/jbc.274.11.6957 pmid:10066749 fatcat:gtge7ci5njhkbjguok6bwmmogm