Two-dimensional agarose gel analysis of simian virus 40 DNA replication intermediates

Abstract

Normal and aberrant simian virus 40 DNA (SV40) replication intermediates can be studied by high-resolution two-dimensional gel electrophoresis. Because the SV40 chromosome is often considered a model for the mammalian replicon, this experimental system can be used to understand how cytotoxic drugs and physical stressors affect DNA replication in mammalian cells. In this article we describe the use of two-dimensional neutral-alkaline and neutral-chloroquine gels in detail. In both systems, first-dimension neutral agarose gels are used to separate viral replication intermediates on the basis of their compactness. The second-dimension gel is then run at a 90° angle to the first. Alkaline second-dimension electrophoresis is carried out in a strongly denaturing, high-pH buffer. Denaturation of nicked or gapped DNA replication intermediates separates the pulse-labeled daughter DNA strands from the unlabeled parental strands. Alkaline second-dimension gels can reveal covalent or topological linkages between parental and daughter DNA strands. In second-dimension chloroquine gel electrophoresis, the electrode buffer contains the intercalating drug chloroquine. Chloroquine titrates out negative supercoils in superhelical DNA replication intermediates and introduces positive supercoils into covalently closed, relaxed intermediates. Because the basis of the separation in each dimension is known, the nature of natural or aberrant DNA replication intermediates can often be deduced from their two-dimensional gel behavior.