Discovery of the Role of Non-B DNA Structures in Mutagenesis and Human Genomic Disorders

Our understanding of the role of non-B DNA structures in mutagenesis and human disease has been an exciting odyssey, but the road traveled has not always been linear or without challenges. Hence, this prologue will provide an overview and some guideposts for the route. Even in my postdoctoral years in the mid-1960s, I realized that DNA sequence was affecting its properties and probably its conformation. Because this concept was heretical at the time and because substantial further work was necessary to cement these concepts, the work moved slowly. I was a postdoctoral fellow with Professor H. Gobind Khorana (Fig. 1) at the Enzyme Institute of the University of Wisconsin-Madison from 1964 to 1966. During that time, the Khorana and Nirenberg teams solved the genetic code, and Gobind shared the Nobel Prize in 1968 with Marshall Nirenberg and Bob Holley (tRNA sequence) for these discoveries. My job as part of this project was to prepare and characterize a number of simple repeating DNA sequences that were subsequently transcribed by me and other workers to provide RNAs of defined repeating sequences. Using these RNAs, we determined the codon assignments; Gobind's brilliance and our hard work made the elucidation of the genetic code (i.e. the language between DNA, RNA, and proteins) straightforward (1). FIGURE 1. Professor H. Gobind Khorana. As part of my postdoctoral studies, I utilized the newly developed Beckman Spinco Model E analytical ultracentrifuge to determine the buoyant densities and other properties of these DNA polymers of repeating sequences (2). From these determinations, there was no doubt that sequences with identical base compositions had very different properties, and further work in my Wisconsin laboratory (described below) during my Assistant and Associate Professorship days verified this conclusion. My laboratory has always been involved in DNA structural problems. However, as opposed to the vast majority of other workers in the field, we were much less interested in small oligonucleotides that can be investigated by x-ray crystallographic or NMR methodologies but instead were interested in large molecules that were in aqueous solution. After all, this was the genuine interface with biology. However, a complication related to this unorthodox interest was that virtually all methodologies for determining structures and their biological functions had to be developed and were not carryovers from other fields (3). Most educators know the old adage that graduate students really do not understand the magnitude of the problems that they are encountering because, if they did, they would never undertake their thesis project. In some ways, I believe that my scientific career adheres to this principle. The 1970s were engaged in biophysical and biochemical investigations on restriction fragments and then recombinant molecules to evaluate the types of non-B DNA structures (described below) that were found at simple repeating sequences. However, a major change occurred in 1990 when I moved to Texas and learned of the involvement of simple repeating triplet sequences in hereditary neurological diseases such as myotonic dystrophy, fragile X syndrome, and Friedreich ataxia. The groundwork laid in Madison and at the University of Alabama at Birmingham (UAB) from the mid-1960s to 1990 was important for elucidating the molecular mechanisms involved in genetic instabilities that are responsible for the etiology of these diseases. Interestingly, the neurological and human genetics communities were quite receptive to the concept of non-B DNA structures (cruciforms, triplexes, slipped structure DNA, quadruplexes, and left-handed Z DNA) (Fig. 2), and thus, major advances were made in the past two decades in the relationship between non-B DNA structures and human diseases. Parenthetically, the term “non-B DNA structures” refers to all DNA conformations other than the orthodox right-handed Watson-Crick structure. FIGURE 2. Non-B DNA conformations involved in genomic disorders. Reprinted with permission from Ref. 56. In the late 1990s and early 2000s, the convergence of bioinformatics, human genetics, molecular biology, and genomics provided powerful new tools for our investigations. In 2004, Albino Bacolla and colleagues discovered that breakpoints for gross deletions that cause a number of human diseases coincide with the presence of non-B DNA structures (described below). These and later studies have cemented the role of non-B DNA structures in human disease. Because of the strength of these techniques and the compelling nature of the results that were obtained, these concepts have been widely accepted and extended by the biological and medical communities. In summary, my teams and I worked for 39 years (1965–2004) to determine the types of non-B DNA structures that exist and to attempt to broach their biological functions. However, my theories and predictions were substantially ahead of the science and methodologies that could be used to rigorously demonstrate these genetic functions. Machiavelli (1513 A.D.) stated, “It must be remembered that there is nothing more difficult to plan, more doubtful of success, nor more dangerous to manage, than the creation of a new system. For the initiator has enmity of all who would profit by preservation of the old institution and merely lukewarm defenders in those who would gain by the new ones.” I certainly agree with Machiavelli but might add that when one turns out to be correct in the end (even if it takes four decades), it is exceedingly rewarding.