Formaldehyde as a probe of DNA structure. r. Mechanism of the initial reaction of Formaldehyde with DNA

McGhee, J.D.; von Hippel, P.H.

Biochemistry 16(15): 3276-3293


ISSN/ISBN: 0006-2960
PMID: 19043
DOI: 10.1021/bi00634a002
Accession: 068518127

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Formaldehyde is used as a probe of the dynamic behavior of native DNA. Initial denaturation rates probably varied with temperature, formaldehyde concentration, DNA melting temperature and DNA MW. EM of DNA from the initial phases of the reaction verifies that denaturation initiates at AT-rich regions in the interior of the DNA molecule. The overall denaturation rate increased with increasing pH. Since the only pH-dependent chemical reaction is at the imino group of thymine (and guanine) located directly in the middle of the Watson-Crick helix, interchain H-bonds probably break prior to reaction. By studying denaturation rates as a function of temperature and pH, under the usual reaction conditions denaturation probably involves adduct formation with the functional groups of thymine and adenine. The thymine reaction (which is rapidly reversible) dominates the denaturation under conditions of high temperature and high pH; conversely, the adenine reaction can be considered to be effectively irreversible and analysis of reaction rates under adenine-reaction-dominated conditions is a vast simplification. By examining reaction rates with single-stranded polynucleotides as a function of temperature, probably must unstack prior to reaction with formaldehyde; following unstacking, the reaction rates are essentially identical to those of mononucleotides. It is also demonstrated that monohydroxymethylated adenine can forn a base pair with thymine, the hydroxymethyl group lying coplanar with the base pair and protruding into the major groove of the double-helical DNA structure. Such a substituted base pair is .apprx. 1.5 kcal/mol less stable than an unreacted AT pair. This stability difference can be quantitatively ascribed to simple stereochemistry and can be used to determine the number of neighboring base pairs which are denatured as a consequence of 1 chemical reaction. The initial reaction of formaldehyde with an adenine moiety in double-helical DNA proceeds as follows: a small sequence of DNA base pairs denatures (i.e., interchain H-bonds break and bases unstack) as a consequence of a local thermal fluctuation; the exocyclic amino group of an adenine residue exposed in this spontaneous fluctuation reacts at the same rate as the free mononucleotide under comparable reaction conditions; and the reacted adenine either re-forms into a (less stable) hydroxymethylated AT base pair, or remains unstacked and unhydrogen bonded, depending on temperature and other environmental factors. Taking these 3 steps into account, reaction rates are predicted from simple helix-coil theory using the experimentally determined loop-weighting functions of Gralla and Crothers. Agreement between calculations and observations is within a factor of 2 for denaturation rates and within .apprx. 1 kcal/mol for apparent activation energies. Conversely, the experimental data can be used to obtain independent estimates of loop-weighting functions describing the behavior of small open loops in DNA, as well as to calculate relative rates of reaction with HCHO at internal nicks and helix ends. The central conclusion from these calculations is that the most probable transiently denatured state of DNA at temperatures below Tm consists of loops containing only 1 open (unstacked and unhydrogen bonded) base pair. The definition, in this series of papers, of the initial reaction mechanism of DNA with formaldehyde should serve as a partial model for the complex molecular pathways involved in processes of genome regulation, such as the interaction of melting proteins with initially native DNA sequences or RNA polymerase with initially closed promoter regions.