Overstretching short DNA – Single-molecule force spectroscopy studies
Structural deformation of DNA has a central role in many biological processes. It occurs, for example, during replication, transcription, and regulation of the activity of the genome. To understand these fundamental processes it is necessary to have a detailed knowledge of the mechanical properties of DNA. How DNA responds to longitudinal stress has been studied on the single-molecule level for over two decades. Early it was discovered that torsionally relaxed double-stranded (ds) DNA undergoes a structural transition when subjected to forces of about 60-70 pN. During this overstretching transition the contour length of the DNA increases by up to 70% without complete strand dissociation. Since its discovery, a debate has arisen as to whether the DNA molecule adopts a new form or denatures under the applied tension. In the work of this thesis the overstretching transition is studied using optical tweezers to extend individual dsDNA molecules of 60 – 122 base pairs. By stretching short designed molecules of variable base-composition and with structural modification, factors determining the outcome of the process could be isolated and investigated. The structural changes induced during the transition vary depending on the stability of the dsDNA. Sequences that have a high GC-content are demonstrated to undergo a reversible overstretching transition into a longer form that remains base-paired. At high salt concentrations, this form of DNA, referred to as S-form, is found to be stable for extended periods of time, while at low salt it quickly denatures. AT-rich sequences are found to denature under tension in two different ways: if the AT-rich domain has one free end, melting will occur by progressive peeling of one strand from the other. When peeling is inhibited, here using synthetic inter-strand crosslinks, melting instead occurs internally within the sequence. The results presented here refine our knowledge of DNA mechanics, essential for understanding how proteins in our cells interact with DNA.
mechanical properties of DNA