Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules

Abstract

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.

Direct Evidence for the Formation of Precatenanes during DNA Replication

“…3, for pBR-terE@StyI the majority of the crossings are expected to be supercoils in the unreplicated region that contains ϳ80% of the molecule. For the same ⌬Lk, the electrophoretic mobility of catenanes would be roughly half that of supercoils (10,63). These RIs migrated very close to unreplicated CCCs (see Fig.…”

Topological patterns in two-dimensional gel electrophoresis of DNA knots

“…This method exploits the empirical observation that the mobility of a charged DNA molecule under an electric field depends on its size, shape, and topology (2). Gel electrophoresis is so reliable that it can be used, for instance, to map replication origins and stalled replication forks (3), to separate plasmids with different amount of supercoiling (3,4), and to identify DNA knots (5,6). The most widely used variant of this technique nowadays is 2D gel electrophoresis, where a DNA molecule is subjected to a sequence of two fields, applied along orthogonal directions (2).…”

“…The most widely used variant of this technique nowadays is 2D gel electrophoresis, where a DNA molecule is subjected to a sequence of two fields, applied along orthogonal directions (2). The two runs are characterized by different field strengths and sometimes also gel concentrations (4); with suitable choices, the joint responses lead to increased sensitivity.…”

“…The terminal velocity can be estimated via a balance between the applied force and the frictional opposing force, which is proportional to the average size of the molecule; as a result, more-complex knots, which are smaller, move faster under the field. However, the mechanisms regulating the electrophoretic mobility of DNA knots at intermediate fields, and in more-concentrated agar gels, are much less understood (4,13,16). Here, experiments suggest that the mobility of knots is usually a nonmonotonic function of the knot complexity, or, more precisely, of their average crossing number (5, 17) (ACN): Initially, knots move more slowly as their ACN increases, whereas, past a critical ACN, more-complex knots move faster.…”

“…Here, experiments suggest that the mobility of knots is usually a nonmonotonic function of the knot complexity, or, more precisely, of their average crossing number (5, 17) (ACN): Initially, knots move more slowly as their ACN increases, whereas, past a critical ACN, more-complex knots move faster. The combination of the responses to external fields directed along two perpendicular directions leads to a characteristic electrophoretic arc, which allows separation of the first simple knots more clearly in a 2D slab (4,6,18,19). To our knowledge, there is currently no theoretical framework that quantitatively explains the nonmonotonic behavior at intermediate or large fields and the consequent formation of arc patterns.…”