Methods to determine DNA structural alterations and genetic instability

Abstract

Chromosomal DNA is a dynamic structure that can adopt a variety of non-canonical (i.e., non-B) conformations. In this regard, at least 10 different forms of non-B DNA conformations have been identified; many of them have been found to be mutagenic, and associated with human disease development. Despite the importance of non-B DNA structures in genetic instability and DNA metabolic processes, mechanisms by which instability occurs remain largely undefined. The purpose of this review is to summarize current methodologies that are used to address questions in the field of non-B DNA structure-induced genetic instability. Advantages and disadvantages of each method will be discussed. A focused effort to further elucidate the mechanisms of non-B DNA-induced genetic instability will lead to a better understanding of how these structure-forming sequences contribute to the development of human disease.

Figure 1

Schematic structure of the mutation-reporter shuttle plasmid, pSP189. Sequences capable of forming non-B DNA structures (shown as “Non-B” in the figure) can be inserted between the supF gene and the promoter located in the amp^R gene.

Figure 2

A. Schematic structure of the mutation reporter shuttle plasmid, pUCNIM. The non-B DNA-forming sequences (shown as “Non-B”) or the control sequence can be inserted between the lacZ gene and the promoter such that the function of the lacZ gene is maintained. B. RT-PCR analysis of dexamethasone-induced transcription through the non-B DNA-forming sequence (CG14) or control sequence in pUCG14 and pUCON plasmids. Primers were designed to bind sequences in the lacZ gene. RT-PCR products were subjected to agarose gel electrophoresis and visualized by ethidium bromide staining.

Figure 3

The YAC breakage assay. Cells containing YAC CF1 (not to scale), which has a non-B DNA-forming sequence (shown as a red star) and URA3 gene, are FOA^S. If breakage occurs in the region around the non-B DNA insertion, the YAC will lose the URA3 gene, resulting in FOA^R. The YAC can be rescued by addition of a yeast telomere onto the C4A4 seed sequence. Adapted from Callahan et al (112).

Figure 4

Schematic structure of the recoverable dual mutation-reporter construct p2RT. The non-B DNA forming sequences (shown as “Non-B”), or the control sequence can be inserted in either the supF gene or the lacZ gene (as shown in figure). The additional LacI binding site (shown as “I” in the figure), and an original one in P_lac are used to pull down the plasmid DNA from the genomic DNA using the LacI protein coupled to magnetic beads. Plasmid DNA can be linearized by EcoRV digestion so that the vector fragment in the genome is flanked by SpeI sites on each side to facilitate isolation of the plasmid DNA from the genomic DNA, and religation for mutation screening.

Figure 5

Schematic diagram of p2RT plasmid rescue from mammalian genomic DNA, and mutation analysis. The detailed procedure is described in the text. “S”: SpeI restriction sites flanking the integrated reporter sequence; LacI binding sites are shown as red triangles; a brown rectangle indicates the lacZ reporter gene, and a gray rectangle indicates the supF reporter gene.