2.7 Summary and Outlook
4.1.1 Distortion of the DNA structure by proteins
In the following section we give a brief overview about some examples of DNA-protein interactions and how they distort the canonical Watson-Crick structure of DNA.
The generic purpose of DNA is to storage the genetic code. The letters of the code are given by a combination of the four nucleotides (or bases) guanine, cytosine and adenine, thymine. Each possible set of three nucleotides in DNA specifies one amino
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Figure 4.1: Schematic representation of the three basic genetic activities in living cells.
a)Transcription: The genetic code is translated into mRNA by RNA Polymerase. b) Replication: Duplication of a dsDNA strand (semiconservative). c) Recombination:
Breakage and reunion of homologous DNA molecules results in chromosomal cross-overs. This results in genetic recombination of alleles. Figures from [10, 118]
acid or a ’stop transcription signal’ for the reading machinery. It enables the physical realization of the genetic code by the synthesis of proteins1. This transcription pro-cess is performed by enzymes, for instance the RNA polymerase in eukaryotic cells.
The basic process is shown in Fig.4.1(a). The enzyme locally melts the Watson-Crick double helix to get access to the nucleotides for copying the sequence into an RNA molecule. With this process the cellular machinery is enabled to express the tran-scribed genes [10]. The progression of E. coli RNA-polymerase on a single dsDNA has been studied by tethering short DNA at one end to a surface and with the other end to a bead. Transcription of DNA by RNA-polymerase influences the fluctuations of the bead characteristically due to change of the length of the DNA. By analyzing these fluctuations the enzymatic activity can be followed [119]. With optical tweezers it was shown that an applied force of 35 pN can stall the work of the DNA polymerase and illustrates nicely the connection between mechanical load and enzymatical activ-ity [120, 121].
For copying the genetic code from cell to cell and from generation to generation DNA
1Viruses are an exception since they have DNA but borrow the reading machinery from their host. If they count to living matter or not is a question of definition.
has to duplicate. This process is called replication, and the scheme of this process is depicted in Fig.4.1(b). First the parental strands have to separate resulting in an Y-shaped replication fork. The unwinding of the double strand is mediated by a class of enzymes called helicase enzymes. Then the two single strands acting as a template for the synthesis of new dsDNA, with the aid of DNA-polymerase. The helicases are also involved in the recombination processes as it is shown in Fig.4.1.c. An example of a helicase which removes the twist in unconstrained DNA is RecBCD. It partic-ipates in the repair of chromosomal DNA through homologous recombination. The translocation of the RecBCD and the unwinding of the double helix can be monitored at single DNA by the release of fluorescent dye molecules during the unwinding pro-cess [122]. Another protein essential for recombination is the RecA protein, which is discussed in more detail in the next section.
Due to the enormously high packaging density of DNA in the cell nucleus many ge-netic reactions as transcription, replication and recombination requires changes in DNA topology [8]. DNA topology is usually described in terms of the linkage number Lk, which is the sum of the numbers T w and W r. The first one called twist counts how often the two strands of the helix winds around each other. For example, uncon-strained DNA in the B-Form has a helix repeat of 10.5 base pairs. If it consists of N base pairs the linkage number is given by Lk = 10.5N . However, in nature circular DNA is generally found to lack one turn of twist for every 17 turns of stable, right handed double helix [10]. This slight underwound of DNA can be compensated by self-intersection of the helix axis. The DNA is called to develop a supercoiled confor-mation2. The strength of the supercoiling is described by the second numberW r the so called writhe. For a constrained DNA as it is the case for circular DNA but also for DNA which is torsionally constrained artificially, the linkage number is a topological constant, i.e. a decrease in the twist causes an increase in the writhe.
A large class of enzymes involved in genetic regulation processes influencing the DNA topology are the topoisomerases. They occur in eucaryotic cells and bacteria as well and are divided in ATP independent (type I) and ATP dependent (type II) types.
The main task of both types is the relaxation of supercoiled helices. They open tem-porally a phosphodiester bond of the DNA followed by an interstrand exchange and reclosing of the phosphodiester bond [8]. The relaxation of the supercoiled DNA was optically observed with the aid of magnetic tweezers [123].
2From daily life everybody knows that due to over- or underwinding of a telephone cable at a certain threshold the cable itself crosses. This is exactly the same what happens in case of DNA.
Figure 4.2: Strand exchange mediated by RecA. From the left-hand side dsDNA spools into RecA/DNA complex. After strand exchange the new dsDNA is released at the right hand side. Figure from [8].
Based on experiments on highly overtwisted DNA and molecular modelling, Allemand et al. [124] suggested that DNA can adopt a structure with 2.6 bases per turn which is 75% longer than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in agreement with Pauling’s early DNA structure [125, 124].