Effect of Hemimethylation and Methylation of Adenine on the Structure and Stability of Model DNA Duplexes

Qiu Guo, Min Lu, Neville R. Kallenbach

Research output: Contribution to journalArticlepeer-review

Abstract

Enzymatic methylation of adenine underlies a variety of biological regulatory mechanisms in Escherichia coli. We present here structural and thermodynamic characterization of a non-selfcomplementary DNA decamer duplex containing the dam sequence 5'-GATC in the unmethylated, hemimethylated (both forms), and methylated states. Differential scanning calorimetry measurements show that the free energies for adenine methylation of the decamer duplex are +1.1 and +2.0 kcal/mol for hemimethylation, respectively, and +3.3 kcal/mol for full methylation. In all cases, a large unfavorable enthalpy change is partially compensated by a favorable entropy term. CD spectroscopy indicates an overall conformational difference between the unmethylated decamer duplex and its methylated analogs. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced in the vicinity of the methylation site of the duplex, consistent with loosening of base pairing at this site. Comparison of the scission patterns of these decamer duplexes by the reactive probes methidiumpropyl- EDTA-FeII [MPE.FeII] and CuI(o-phenanthroline)2 [(OP2CuI] indicates that the methylation site of the decamer duplex represents a site of enhanced reactivity for these agents. On the basis of these thermodynamics and structural features, we suggest that the methylated base pair exists in two different helical states, which require local transient opening of the duplex for interconversion.

Original languageEnglish (US)
Pages (from-to)16359-16364
Number of pages6
JournalBiochemistry
Volume34
Issue number50
DOIs
StatePublished - Dec 1995

ASJC Scopus subject areas

  • Biochemistry

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