Intervening DNA

The second NIST human DNA SRM is a PCR-based DNA Profiling Standard. The PCR was first described by Saiki et al. (1985,1989). Since then it has developed into a highly versatile and widely used detection, identification, manipulation and analysis tool in molecular biology, including DNA profiling. In brief, two short synthetic oligonucleotides, or primers, are used to define an intervening DNA sequence... 

In order to directly probe the dynamics of CT between Et and ZG, and to understand how the intervening DNA base stack regulates CT rate constants and efficiencies, we examined this reaction on the femtosecond time scale [96]. These investigations revealed not only the unique ability of the DNA n-stack to mediate CT, but also the remarkable capacity of dynamical motions to modulate CT efficiency. Ultrafast CT between tethered, intercalated Et and ZG was observed with two time constants, 5 and 75 ps, both of which were essentially independent of distance over the 10-17 A examined. Significantly, both time constants correspond to CT reactions, as these fast decay components were not detected in analogous duplexes where the ZG was re-... 

DNA CT also permits chemistry at a distance. Oxidative DNA damage and thymine dimer repair can proceed in a DNA-mediated reaction initiated from a remote site. These reactions too are sensitive to intervening DNA dynamical structure, and such structures can serve to modulate DNA CT chemistry. The sensitivity of DNA CT to base pair stacking also provides the basis for the design of new DNA diagnostics, tools to detect mutations in DNA and to probe protein-DNA interactions. 

This reduction is achieved through meiosis, a process in which two successive ceU divisions occur without intervening DNA duplication. As with the cell cycle, DNA duphcation wiU have aheady taken place prior to the cell division, so that during the initial prophase each pair of identical DNA molecules forms a pair of chromatids attached to each other at a centrosome. In contrast to mitosis, however, homologous chromosomes now pair up (metaphase I). Note that in a pair of homologous chromosomes each wiU contain the same genes (one of the pair having come from the mother and one from the father) but many of the alleles wiU be different. 

Fig. 1.19. Tetramerization of the Lac repressor and loop formation of the DNA. The Lac repressor from E. coli binds as a dimer to the two-fold symmetric operator seqnence, whereby each of the monomers contacts a half-site of a recognition sequence. The Lac operon of E. coli possesses three operator sequences Of, 02 and 03, aU three of which are required for complete repression. Of and 03 are separated by 93 bp, and only these two sequences are displayed in the figure above. Between Of and 03 is a binding site for the CAP protein and the contact surface for the RNA polymerase. The Lac repressor acts as a tetramer. It is therefore assumed that two dimers of the repressor associate to form the active tetramer, whereby one of the two dimers is bound to 03, the other dimer binds to Of. The intervening DNA forms a so-caUed repression loop. After Lewis et al., 1996.

This type of promoter displays markedly different characteristics compared to the o -dependent promoter. The o -contammg holoenzyme binds tightly to the promoter in the absence of transcriptional activators. In this closed state, however, it is not capable of initiating transcription. The transcriptional activators are required in this case to activate the promoter-bound holoenzyme for initiation, i.e. to transform it into the open complex (see Fig. 1.29). Activation is mediated via protein-protein interactions between the transcriptional activator and the RNA polymerase holoenzyme, and is accompanied by ATP hydrolysis. The binding site for the transcriptional activator is found at a distance of ca.llO bp upstream form the start site and can be shifted further upstream without loss of stimulatory effect. Direct interaction of the holoenzyme with the bound transcriptional activator is possible due to loop formation of the intervening DNA. The strict dependency on transcriptional activators for transcription initiation indicates that the DNA-bound holoenzyme alone is not capable of isomerizing to the transcription-competent open complex. The transition to the open complex requires interactions with the transcriptional activator, an event which occurs with ATP hydrolysis. 

Fig. 1.29. Mechanism of promoter activation of (/ -dependent genes in procaryotes. The formation of an open, initiation-competent transcription complex for (/ -dependent genes requires the assistance of transcription activators, which bind to their cognate UAS element. Upon loop formation of the intervening DNA sequences, the transcription activator interacts with the (/ -con-taing RNA polymerase bound to the promoter. The activation is accompanied by ATP hydrolysis and leads to the formation of an open complex.

The sequences of the recombination sites recognized by site-specific recombinases are partially asymmetric (nonpalindromic), and the two recombining sites align in the same orientation during the recombinase reaction. The outcome depends on the location and orientation of the recombination sites (Fig. 25-39). If the two sites are on the same DNA molecule, the reaction either inverts or deletes the intervening DNA, determined by whether the recombination sites have the opposite or the same... 

As a stem cell in the bone marrow differentiates to form a mature B lymphocyte, one V segment and one J segment are brought together by a specialized recombination system (Fig. 25-44). During this programmed DNA deletion, the intervening DNA is discarded. There are about 300 X 4 = 1,200 possible V-J combinations. 

If recombination occurs within a piece of DNA at two homologous sites such as the attL and attR sites at the boundaries of the X prophage, the intervening DNA will be excised as a circular particle (Eq. 27-15). In this instance the two homologous regions must be repeated in the same direction, as is indicated by the arrow structures in Eq. 27-15. If the homologous sequences are oriented in opposite directions, i.e., they are inverted repeats, excision will not occur but the piece of DNA between the repeats will be inverted (Eq. 27-16). 

Fig. 1. In the germ-line (embryo) DNA, sequences coding for the variable (V) region lie distant from those encoding the constant (C) region. During the differentiation of B lymphocytes, these two sequences are brought together to form an active antibody gene by deletion of the intervening DNA (somatic recombination).

From spectroscopic and biochemical studies it has become clear that DNA-mediated CT is extremely sensitive to the re-stacking of the intervening DNA bases and to disruption and perturbation of the DNA structure or conformation. This indicates that sensing of DNA damage could be accomplished, at least in part, on the basis of CT chemistry. In considering these possibilities, it is important to discover whether DNA-mediated CT does occur within the cell. Charge transfer in HeLa cell nuclei has recently been probed by use of a rhodium photooxidant [15]. 

It is thought that enhancers function by assembling a complex of transcription factors that interact with the proteins and the promoter in such a way that the intervening DNA is looped out . 

Figure 33.15. VJ Recombination. A single V gene (in this case, V2) is linked to a J gene (here, J4) to form an intact VJ region. The intervening DNA is released in a circular form. Because the V and J regions are selected at random and the joint between them is not always in exactly the same place, many VJ combinations can be generated by this process.

Transcription factors can often act even if their binding sites lie at a considerable distance from the promoter. These distant regulatory sites are called enhancers (p. 838). The intervening DNA can form loops that bring the enhancer-bound activator to the promoter site, where it can act on other transcription factors or on RNA polymerase. 

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