Structure of branched DNA molecules: Gel retardation and atomic force microscopy studies

Elena A. Oussatcheva, Luda S. Shlyakhtenko, Ross Glass, Richard R. Sinden, Yuri L. Lyubchenko, Vladimir N. Potaman

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.

Original languageEnglish (US)
Pages (from-to)75-86
Number of pages12
JournalJournal of Molecular Biology
Volume292
Issue number1
DOIs
StatePublished - Sep 10 1999

Keywords

  • Atomic force microscopy
  • Bending
  • Electrophoresis
  • Heteroduplex DNA
  • Isomers

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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