Deletions of bases in one strand of duplex DNA, in contrast to single-base mismatches, produce highly kinked molecules: possible relevance to the folding of single-stranded nucleic acids.

Abstract

A 32-base-pair (bp) DNA duplex with deletions in one strand, and thus extra bases in the opposing strand, was ligated head-to-tail to produce linear and circular multimers. The electrophoretic mobility of the linear multimers was analyzed in polyacrylamide gels and the size of the circular DNA was determined by electron microscopy. A 1-base deletion produced a marked retardation in the mobility of the linear multimers coincident with the formation of a population of multimeric circles of a smaller average size than the deletionless 32-mer; 2-, 3-, or 4-base deletions at the same site produced proportionately greater effects. Two 1-base deletions separated by 10 bp on the same strand produced a greater reduction in mobility than a 1-base deletion, whereas two 1-base deletions spaced by 5 bp on the same strand yielded a molecule that behaved more like the deletionless DNA. We conclude that deletions of 1-4 bases at a single site on duplex DNA produce molecules that behave as if they contain sharp bends or kinks. In contrast, single mismatches in the 32-bp duplex produced no abnormality in behavior relative to normally base-paired DNA in the gel mobility and electron microscopic assays. The possible role of such structures in organizing the three-dimensional folding of single-stranded nucleic acids is considered.Extra bases in one strand of double-stranded DNA or RNA may result from a variety of natural mechanisms. When single-stranded RNA or DNA folds on itself, the resulting secondary structure can be thought of as a chain of duplex rods containing perfectly and imperfectly paired bases linked by junctions containing extra bases in one of the two opposing strands. How these junctions contribute to the overall folding of single-stranded DNA or RNA is not established, but were they to provide a precise orientation to the adjoining duplex segments, a possibility raised here, an understanding of their structure would be very important-in particular to our knowledge of the folding of natural RNA.Extra or deleted bases can also be found in duplex DNA as a result of recombination or of errors in replication. In vitro, the RecA protein of Escherichia coli readily catalyzes recombination reactions between two DNAs that differ from each other by a number of bases, giving rise to recombinant heteroduplexes containing base deletions in one strand (1, 2). In vitro studies have suggested how the deletion of bases during replication could lead to frameshift mutations (3), and in vivo, bacteriophage T4 DNA polymerase plays a central role in controlling the frequency of frameshift mutations (4). Deletions or insertions of bases in one strand can result from DNA synthesis in vitro where, in general, single-base deletions predominate and most often occur in runs of the same nucleotide (5-7). In E. coli, the repair of deletions of up to 10 bases in one strand of DNA is carried out by the methyldirected repair system, which also corrects mismatches in DNA (ref. 8, reviewed in ref. 9). To understand how base deletions or inser...

Sequencing-Based Detection of Low-Frequency Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by an RNA/DNA Heteroduplex Generator-Tracking Assay

“…When RNA/DNA heteroduplexes are generated with patients' protease variants, the six extra inserted nucleotides in the RNA strand are accommodated by looping them out of the heteroduplex, causing a highly kinked double-stranded structure whose electrophoretic mobility through polyacrylamide is greatly retarded (Fig. 1B) (39). The exact structure of the heteroduplex molecule at each of the nucleotide insertion sites is highly dependent on the regional nucleotide sequence.…”

Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions.

“…The electrophoretic mobility of such heteroduplexes should be dependent on the portion of singlestranded sequence present in the structure. (14) Heteroduplexes with a small number of sequence mismatches will contain only a small portion of singlestranded DNA and are expected to run slightly slower than their corresponding homoduplexes. Heteroduplexes, with composition >50% single-stranded DNA, would be expected to move more slowly than the corresponding single-stranded structures that make up the heteroduplex.…”

A loop–loop interaction and a K-turn motif located in the lysine aptamer domain are important for the riboswitch gene regulation control

“…The gel electrophoresis technique is very sensitive to study global conformational changes in nucleic acids (Marini et al 1982;Diekmann and Wang 1985;Bhattacharyya and Lilley 1989;Hsieh and Griffith 1989;Bhattacharyya et al 1990;Tang and Draper 1990;Luebke and Tinoco 1996;Lafontaine et al 2001Lafontaine et al , 2002aGoody et al 2004;Lemay et al 2006;Lemay and Lafontaine 2007), and provides a simple yet powerful means to analyze the K-turn motif in solution. To analyze the extent of bending, a set of comparison duplexes was constructed by placing a series of oligoadenine bulges in the same sequence context, to generate RNA duplexes having the same length as the K-turn-containing molecules.…”